The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli.
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 DNA stimulates OmpR phosphorylation by EnvZ [Ames, S. K., Frankema, N. & Kenney, L. J. (1999) Proc. Natl. Acad. Sci. USA 96, 11792-11797]. 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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Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Yoshida
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Inouye
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D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; D011494:Protein Kinases; C057843:envZ protein, E coli