Enteric bacteria produce a set of proteins, termed porins, that act as passive diffusion pores to allow small hydrophilic molecules to cross the outer membrane permeability barrier. Escherichia coli K12 contains two major, non-specific porins, OmpF and OmpC. In general, the total amount of these two porins present in the cell is constant. However, the relative levels fluctuate in response to a variety of environmental conditions. One environmental factor that appears to be particularly important is media osmolarity. If osmolarity is high, OmpC predominates. Conversely, in dilute media, OmpF is found. It is thought that by sensing osmolarity, E. coli can distinguish its common habitats, i.e., inside or outside of a host animal. We wish to understand the molecular basis for this regulatory system. Previous work from our lab has identified two genes, ompR and envZ, which specify proteins that are required to activate porin gene expression. EnvZ appears to function as a membrane-bound signal transducer to monitor the environment and direct the effector protein, OmpR (a DNA-binding protein) to the appropriate cis-acting sites at the ompF and ompC promoters. OmpR and EnvZ share significant homology with the sensor and effector proteins of other two-component regulatory systems found in a variety of organisms. The structure and function of OmpR will be probed through a detailed genetic and biochemical characterization of 70 ompR missense mutations which confer a variety of porin phenotypes. Results obtained should clarify the nature of the genetic switch that controls porin fluctuation and assign roles to each of the multiple cis-acting sites at the ompF and ompC promoters. Additional experiments seek to verify the existence of an interaction between OmpR and the a subunit of RNA polymerase and to probe its mechanistic implications. These studies address the question of how positive activators, such as OmpR, function to stimulate transcription. Lastly, a detailed genetic analysis of envZ is planned which is similar to, and will complement, our genetic analysis of ompR. The goal of this analysis is to gain insights into the sensory functions of EnvZ and how it acts mechanistically as a signal transducer to throw the genetic switch.
Showing the most recent 10 out of 28 publications
