In prokaryotic cells adaptive responses to environmental change can be mediated by a conserved family of modulator and effector molecules which include genes that regulate bacterial pathogenesis. The communication between the modulator and effector proteins can occur via a novel phosphotransfer reaction. The objective of this proposal is to elucidate the molecular mechanisms by which these conserved proteins control environmentally regulated gene expression in bacteria. In E. coli an inner membrane protein, EnvZ, undergoes autophosphorylation at a histidine residue and transfers its phosphate moiety to a carboxyl group on the transcription activator (OmpR). OmpR in turn regulates the expression of the genes that encode the outer membrane porin proteins, OmpF and OmpC. Using parent as well as ompR and envZ mutant strains of E. coli the underlying molecular details of how the phosphorylated state, DNA-binding properties and the transcriptional regulatory activities of OmpR control the adaptive responses to changes in the osmolarity of the growth medium will be examined. This will be accomplished by use of the following methods; in vivo and in vitro phosphorylation assays, gel retardation and hydroxyl radical footprinting approaches, Western immunoblotting techniques, PCR amplification of envZ alleles, site-directed mutagenesis and in vitro transcription assays. In addition, the biochemical characterization of the site(s) of phosphorylation of EnvZ and OmpR, and the analysis of the domain structure of OmpR are planned. For the latter study a hybrid protein containing the phosphoacceptor domain of the chemotaxis effector molecule Che Y, and the DNA-binding domain of OmpR will be constructed. Since recent findings indicate that proteins that are homologous to OmpR and EnvZ regulate the expression of virulence genes in several bacteria, it is likely that the molecular understanding of environmentally regulated gene expression in E. coli will provide new insights for understanding the molecular events of bacterial pathogenesis.
