Abstract
OmpR is the response regulator of a two-component regulatory system that controls the expression of the porin genes ompF and ompC in Escherichia coli. This regulator consists of two domains joined by a flexible linker region. The amino-terminal domain is phosphorylated by the sensor kinase EnvZ, and the carboxyl-terminal domain binds DNA via a winged helix-turn-helix motif. In vitro studies have shown that amino-terminal phosphorylation enhances the DNA binding affinity of OmpR and, conversely, that DNA binding by the carboxyl terminus increases OmpR phosphorylation. In the present work, we demonstrate that the linker region contributes to this communication between the two domains of OmpR. Changing the specific amino acid composition of the linker alters OmpR function, as does increasing or decreasing its length. Three linker mutants give rise to an OmpF(+) OmpC(-) phenotype, but the defects are not due to a shared molecular mechanism. Currently, functional homology between response regulators is predicted based on similarities in the amino and carboxyl-terminal domains. The results presented here indicate that linker length and composition should also be considered. Furthermore, classification of response regulators in the same subfamily does not necessarily imply that they share a common response mechanism.
Highlights
Two-component signaling systems are the predominant signal transduction pathways in prokaryotes, and the components are highly conserved [1]
In Escherichia coli, a two-component system that consists of the sensor kinase EnvZ and the response regulator OmpR regulates the expression of the outer membrane porins OmpF and OmpC in response to environmental osmolarity
We discovered that a linker of 13–15 amino acid residues is optimal for OmpR function, with shorter linkers gradually decreasing the ability of the protein to activate transcription of the porin genes (Fig. 2)
Summary
As the putative interdomain interface might vary with linker length, it is useful to understand the required length of the linker region for each regulator. We express OmpR constructs encoding linkers of various lengths and find that alterations in linker length impair OmpR function. In our analysis of several linker mutants, it was striking that three different substitutions resulted in an OmpFϩ OmpCϪ phenotype. An examination of these OmpR mutants revealed that their porin gene expression profiles are similar, and yet they differ in their phosphorylation and DNA binding properties. There are different molecular mechanisms by which an OmpR linker mutant may present an OmpFϩ OmpCϪ phenotype in vivo
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