Hierarchical and co‐operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli

Bergstrom, Lisa C.; Qin, Ling; Harlocker, Susan L.; Egger, Linda A.; Inouye, Masayori

doi:10.1046/j.1365-2443.1998.00228.x

Cited by 33 publications

(42 citation statements)

References 46 publications

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“…2, 3, 5, and 6). It is not yet understood how OmpR regulates ompF and ompC expression (7)(8)(9)(10)(11), although it appears that at low concentrations of OmpR-P, ompF is activated, whereas at high concentrations of OmpR-P, ompF is repressed and ompC is activated (12)(13)(14).…”

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confidence: 99%

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

Batchelor

Goulian

2003

Proc. Natl. Acad. Sci. U.S.A.

227

308

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The EnvZ͞OmpR system in Escherichia coli, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling. Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR. We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation. The model predicts that when EnvZ is much less abundant than OmpR, as is the case in E. coli, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ. The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high. To test these predictions, we have perturbed the porin regulatory circuit in E. coli by varying the expression levels of EnvZ and OmpR. We have constructed two-color fluorescent reporter strains in which ompF and ompC transcription can be easily measured in the same culture. Using these strains we have shown that, consistent with the predictions of our model, the transcription of ompC and ompF is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.A mong prokaryotes, a remarkable number of cellular functions are controlled by two-component regulatory systems. Dedicated circuits transduce and interpret specific signals such as pH, temperature, osmolarity, light, nutrients, ions, pheromones, and toxins to regulate a wide range of processes including motility, virulence, metabolism, the cell cycle, developmental switches, antibiotic resistance, and stress responses (for reviews see for example refs. 1-4). Most of the detailed features of these circuits are quite varied and depend on the particular signals that are detected and the responses that are effected. However, all twocomponent systems transmit information via phosphorylation of a histidine on one protein (a histidine kinase) followed by transfer of phosphate to an aspartic acid, which is usually on a distinct protein (a response regulator). A notable feature of many of these systems is that the histidine kinase is bifunctional: it phosphorylates and dephosphorylates its cognate response regulator. This feature raises the interesting question of what is gained by implementing a regulatory circuit in this manner. Would a circuit in which, for example, phosphorylation is controlled by the histidine kinase and dephosphorylation is caused by spontaneous hydrolysis or a separate phosphatase have important differences from a circuit in which both reactions are mediated by the same enzyme?A particularly well-characterized example of a bifunctional histidine kinase is EnvZ. In Escherichia coli, EnvZ responds to changes in the extracellular osmolarity of inner-membrane impermeable compounds and controls the phosphorylation of the response regulator OmpR. OmpR, in turn, regulates the transcription of a number of genes, the best characterized of which are ompF a...

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mentioning

confidence: 99%

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

Batchelor

Goulian

2003

Proc. Natl. Acad. Sci. U.S.A.

227

308

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“…As shown in Fig. 3B, the patterns of OmpR-P formation with DNA fragments that cannot bind OmpR-P independently, including F2 (lanes 5-8), F3 (lanes 9-12), F4 (lanes [17][18][19][20], and C1(C5G) (lanes [21][22][23][24], are almost identical to those with F1 (lanes 1-4) and C1 (lanes 13-16), which bind OmpR-P. These results indicate that DNA has no effect on the EnvZcT247Y's OmpR kinase activity that consists of autophosphorylation and phosphotransfer reactions.…”

Section: Resultsmentioning

confidence: 99%

“…The mechanism of the reciprocal transcription of ompF and ompC mediated by OmpR-P has been proposed (21). At low osmolarity, OmpR-P cooperatively binds to F1 as well as F2 and somewhat loosely to F3 resulting in ompF expression by direct interaction of bound OmpR-P with the C-terminal domain of the ␣ subunit of RNA polymerase (29,30), whereas ompC is not induced, because OmpR-P is able to bind only to C1 but not to C2 and C3 sites.…”

Section: Discussionmentioning

confidence: 99%

“…The phosphorylation of OmpR at Asp-55 enhances its ability to bind to the regulatory sequences upstream of the ompC and ompF promoters (16,17). In vitro and in vivo footprinting studies have shown that OmpR-P binds to the Ϫ380 to Ϫ361 region (F4 site), the Ϫ100 to Ϫ39 region (F1, F2, and F3 sites) of ompF, and the Ϫ100 to Ϫ38 region (C1, C2, and C3 sites) of ompC (18)(19)(20)(21). These binding sites consist of 20 base pairs each and share a consensus sequence (22).…”

mentioning

confidence: 99%

“…Two OmpR-P molecules bind to each site in a head-to-tail manner (23). The binding affinities of OmpR-P to these sites are in a hierarchical order such that OmpR-P binds independently to F1 and C1 sites and bind to other sites only after the upstream F1 or C1 site is occupied (17,21).…”

mentioning

confidence: 99%

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The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

Qin¹

2001

Proceedings of the National Academy of Sciences

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Escherichia coli modulates its porin expression through a histidine kinase, EnvZ, and its cognate response regulator, OmpR. EnvZ is a bifunctional enzyme that possesses both OmpR kinase and phosphorylated OmpR (OmpR-P) phosphatase activities and thus controls the cellular level of OmpR-P. In an in vitro-assay system, the addition of OmpR to the reaction mixture consisting of the cytoplasmic domain of EnvZ (EnvZc) and ATP produces a barely detectable amount of OmpR-P because of the dual activities of EnvZ. Here we report that DNA fragments containing the upstream promoter regions of the porin genes (ompF and ompC) can shift the equilibrium between OmpR and OmpR-P dramatically toward OmpR-P. Among the four reactions occurring in the mixture, only the EnvZ phosphatase activity was inhibited severely by the specific DNA, in contrast to the previous report by Kenney and her associates that The autophosphorylation of EnvZc and the phosphotransfer from phosphorylated EnvZc to OmpR were not affected by DNA, whereas the autodephosphorylation of OmpR-P was inhibited slightly. We propose that the apparent inhibitory effect of DNA on the EnvZ phosphatase function is caused by sequestrating OmpR-P from the reaction as a result of OmpR-P binding to DNA.

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Identification of Regulatory Sequences and Expression Analysis of OmpR Gene Under Different Stress Conditions in the Antarctic Bacterium Psychrobacter sp. G

2012

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An OmpR gene, named OmpR503, was cloned from the Antarctic psychrotrophic bacterium Psychrobacter sp. G according to its genomic draft. The deduced amino acid sequences of OmpR503 were highly conserved with other known protein members of OmpR family. qRT-PCR analysis showed that the expression of OmpR503 gene was significantly enhanced by high salinity (90, 120). The expression of OmpR503 gene was also significantly increased at low temperature (0, 10 °C), whereas depressed at high temperature (30 °C). When the strain was subjected to combined stress (0 °C with a salinity of 90), the expression of OmpR503 gene was increased significantly, which was up to 3.0-fold. In Antarctica, freezing tolerance of psychrotrophic bacteria is often accompanied by tolerance to osmotic stress caused by a lack of free water, thus the cold inducibility of OmpR503 gene might help the strain adapt to the harsh environment more efficiently.

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Hierarchical and co‐operative binding of OmpR to a fusion construct containing the ompC and ompF upstream regulatory sequences of Escherichia coli

Cited by 33 publications

References 46 publications

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

Identification of Regulatory Sequences and Expression Analysis of OmpR Gene Under Different Stress Conditions in the Antarctic Bacterium Psychrobacter sp. G

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