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J. Biol. Chem., Vol. 276, Issue 44, 40896-40902, November 2, 2001

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K⁺ Stimulates Specifically the Autokinase Activity of Purified and Reconstituted EnvZ of Escherichia coli^*

Kirsten Jung, Knut Hamann, and Anne Revermann

From the Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany

The histidine kinase/response regulator system EnvZ/OmpR of Escherichia coli regulates transcription of the genes ompF and ompC, encoding two porins of the outer membrane. Although the total amount of OmpF and OmpC remains constant, the relative levels of the two proteins fluctuate in a reciprocal manner depending on medium osmolality. The membrane-anchored sensor EnvZ somehow monitors changes in environmental osmolality. To characterize the nature of the stimulus perceived by EnvZ, this protein was overproduced, purified, and reconstituted into proteoliposomes. Autokinase activity of purified and reconstituted EnvZ was stimulated by an increase of the K⁺ concentration. Rb⁺, Na⁺, and NH also stimulated the activity but to a smaller extent, whereas an osmotic upshift imposed by various sugars or increasing concentrations of glycine betaine, proline, or Tris/MES were without influence. Neither the transfer of the phosphoryl group from EnvZ~P to OmpR nor the EnvZ-mediated OmpR~P dephosphorylation were affected by one of the tested solutes. Experiments with the reconstructed signal transduction cascade including DNA fragments demonstrated a substantial increase of the amount of phosphorylated OmpR in the presence of K⁺ and to a lower extent in the presence of Na⁺, Rb⁺, and NH. Various K⁺ salts were tested indicating that the determined effects were K⁺-specific and not dependent on the anion. In a further in vitro test system, which utilizes right-side-out membrane vesicles, the K⁺-specific activation of EnvZ autokinase from the luminal side was confirmed. These results clearly indicate a regulation of EnvZ autokinase activity by monovalent ions, specifically K⁺. Whether K⁺ accumulation, which is one of the first responses of E. coli after an osmotic upshift, is related to the stimulation of the EnvZ autokinase activity in vivo is discussed.

This article is dedicated to Karlheinz Altendorf (Osnabrück) on the occasion of his 60th birthday.

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