Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR

Abstract

The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions. Expression of the major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12 is regulated in response to a wide variety of environmental signals (reviewed in refs. 1-3). The most extensively studied of these signals is the osmolarity of the growth medium. At low osmolarity, the OmpF porin is preferentially expressed and only low levels of the OmpC porin are found. Conversely, at high osmolarity, OmpC is preferentially expressed and only low levels of OmpF are found. This fluctuation in porin expression is controlled at the transcriptional level by the two-component regulatory system, EnvZ and OmpR. EnvZ is a membrane-bound histidine kinase that modulates the activity of the DNA-binding protein OmpR via phosphorylation (refs. 2 and 3 and references therein). The phosphorylated form of OmpR (OmpR-P), which is the active form of the protein, is a transcriptional activator at the ompC promoter and can function as either an activator or a repressor at the ompF promoter (4).According to the current model (3), at low osmolarity the cellular concentration of OmpR-P is low. This level is sufficient for OmpR-P to interact with the sites in the ompF regulatory region that are responsible for activating ompF transcription. At high osmolarity, the cellular concentration of OmpR-P is much greater. As a result of this increase, OmpR-P is now capable of occupying the sites in the ompC regulatory region responsible for activating ompC transcription, and the sites at ompF responsible for repressing ompF transcription. Thus, this model predicts that the regulation of ompF and ompC is a direct consequence of the level of OmpR-P in the cell and is dependent on the way in which OmpR-P interacts with sites in the ompF and ompC regulatory regions.A number of studies have addressed the basi...

Sites internal to the coding regions of phoA and pstS bind PhoP and are required for full promoter activity

“…In all cases, the phosphorylation of response regulators has been reported to affect their binding to the target promoters, by increasing binding affinity, such as OmpR and PhoB (Aiba et al, 1989;, by changing the DNA binding pattern, such as UhpA (Dahn et al, 1997;Boucher et al, 1997), or by stimulating co-operative binding to DNA, such as NtrC (Porter et al, 1993;Huang et al, 1997), to activate gene transcription. The binding pattern of PhoP to the core binding regions of the pstS and phoA promoters shows similarities to that of UhpA binding to the uhpT promoter, in which there are high-and low-affinity binding sites for the unphosphorylated form of response regulator.…”

Characterization of Virulence Factor Regulation by SrrAB, a Two-Component System in Staphylococcus aureus

“…The identification of multiple shifted bands in these gel shift assays was not surprising, as gel shift studies with OmpR and SarA revealed multiple shifted bands at target promoters (14,27). An identical gel shift reaction with 100 bp of the S. aureus ribosomal protein rpsC did not produce a shifted band, indicating that the SrrA binding to agr, spa, srr, and tst promoters is not due to nonspecific association with staphylococcal DNA.…”

Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

“…The nonphosphorylated form of StyR was shown to be a monomer, but on phosphorylation the two monomers join to form a dimer (124). This is also known to occur in other two-component regulatory systems (37,64,89,129,243). StyR in dimeric form has a 10-fold-higher affinity for the styA promoter than does the monomer (124), which could mean that dimerization of the response regulator is needed for efficient recruitment of the protein on the promoter and subsequent transcription activation.…”