A single amino acid substitution in the C terminus of OmpR alters DNA recognition and phosphorylation

In bacteria and lower eukaryotes, adaptation to changes in the environment is often mediated by two-component regulatory systems. Such systems provide the basis for chemotaxis, nitrogen and phosphate regulation and adaptation to osmotic stress, for example. in Escherichia coli, the sensor kinase Env Z detects a change in the osmotic environment and phosphorylates the respon se regulator OmpR. Phospho-OmpR binds to the regulatory regions of the pori n genes ompF and ompC, and alters their expression. Recent evidence suggest s that OmpR functions as a global regulator, regulating additional genes be sides the porin genes. Ln this study, we have characterized a previously is olated OmpR2 mutant (V203M) that constitutively activates ompF and fails to express ompC. Because the substitution was located in the C-terminal DNA-b inding domain, it had been assumed that the substitution would not affect p hosphorylation of the N-terminal domain of OmpR. Our results indicate that this substitution completely eliminates phosphorylation by a small phosphat e donor, acetyl phosphate, but not phosphorylation by the kinase EnvZ. The mutant OmpR has altered dephosphorylation kinetics and altered binding affi nities to both ompF and ompC sites compared to the wild-type. Thus, a singl e amino acid substitution in the C-terminal DNA-binding domain has dramatic effects on the N-terminal phosphorylation domain. Most strikingly, we have identified a single base change in the OmpR binding site of ompC that rest ores high-affinity binding activity by the mutant. We interpret our results in the context of a model for porin gene expression. (C) 2000 Academic Pre ss.