K+ stimulates specifically the autokinase activity of purified and reconstituted EnvZ of Escherichia coli

The histidine kinase/response regulator system EnvZ/ OmpR of Escherichia co li regulates transcription of the genes ompF and ompC, encoding two porins of the outer membrane. Although the total amount of OmpF and OmpC remains c onstant, the relative levels of the two proteins fluctuate in a reciprocal manner depending on medium osmolality. The membrane-anchored sensor EnvZ so mehow monitors changes in environmental osmolality. To characterize the nat ure of the stimulus perceived by EnvZ, this protein was overproduced, purif ied, and reconstituted into proteoliposomes. Autokinase activity of purifie d and reconstituted EnvZ was stimulated by an increase of the K+ concentrat ion. Rb+, Na+, and NH4+ also stimulated the activity but to a smaller exten t, whereas an osmotic upshift imposed by various sugars or increasing conce ntrations of glycine betaine, proline, or Tris/MES were without influence. Neither the transfer of the phosphoryl group from EnvZ similar toP to OmpR nor the EnvZ-mediated OmpR similar toP dephosphorylation were affected by o ne of the tested solutes. Experiments with the reconstructed signal transdu ction cascade including DNA fragments demonstrated a substantial increase o f the amount of phosphorylated OmpR in the presence of K+ and to a lower ex tent in the presence of Na+, Rb+, and NH4+. Various K+ salts were tested in dicating 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+ ac cumulation, 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.