Two-component regulatory systems represent the major paradigm for signal transduction in prokaryotes. The simplest systems are comprised of a sensor kinase and a response regulator. The system being studied consists of EnvZ, the sensor and OmpR, the response regulator. The prevailing view is that OmpR regulates the porin genes in response to changes in osmolarity of the growth medium. At low osmolarity, OmpF is the major porin in the outer membrane and at high osmolarity, ompF is repressed and OmpC is the major porin in the outer membrane. Sensing the environment and regulating the porins is one way that E. coli determines whether it is in a host intestine or out in the pond. The long-held view of porin regulation is being called into question by recent measurements of ompC transcription in an array of E. coli strains. Previous measurements were performed on a strain of E. coli that contained very large deletions compared to the sequenced strain. Using new real-time RT-PCR technology, a level of post-transcriptional regulation was discovered, representing a paradigm shift in terms of regulation by this well-characterized two-component system. These new findings emphasize the need for a systematic re-examination of the biochemical properties and partial reactions that underlie regulation of the porin genes (Objective b). Experiments are designed to elucidate the mechanism and determine whether regulation is translational or at the level of protein turnover. In the most recent funding period, unique cysteine substitutions were employed in a cross-linking study to examine the orientation of OmpR at ompF and ompC sites. It was discovered that OmpR adopts a new conformation when binding to DNA ("head-to-head") rather than the previously suggested mode ("head-to-tail"). These studies suggest that amino acids of OmpR contact DNA via an alternate helix than previously believed and experiments are proposed to test this hypothesis (Objective a). OmpR is a global regulatory protein, regulating many genes in addition to the porin genes. Understanding how OmpR functions as a transcriptional activator and repressor, as well as the cues that it responds to will aid in understanding signal transduction in general as well as how OmpR regulates expression of virulence genes in many pathogens. Broader impacts of this project include participation of undergraduate students and representatives of underrepresented minorities in the research group, teaching courses in microbiology in Mexico and Chile, and joining international efforts to recruit students to careers in biophysics.
