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, then 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 out of a host animal. We wish to understand the molecular basis for this regulatory system. Previous work from our lab and others have identified two regulatory genes, ompR and envZ, which comprise an operon, ompB. These two genes specify proteins that are required to activate porin gene expression. Several ompR-lacZ fusions that specify hybrid proteins with partial OmpR activities have been isolated. Selections that exploit these bifunctional fusions have been designed and will be employed for a detailed structure/function analysis of OmpR. A similar strategy will be used with EnvZ. To study protein-protein interactions and to analyze the cis-acting target sites of the regulatory proteins, second-site suppressor mutations will be isolated and characterized by in vitro assay and DNA sequence analysis. Finally, the generality of osmoregulation will be investigated by isolating and characterizing a large and representative series of lacZ fusions to osmoregulated genes.
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