Abstract
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 NH4(+) 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 approximately P to OmpR nor the EnvZ-mediated OmpR approximately 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 NH4(+). 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.
Highlights
Tor OmpR expression of ompC and ompF, encoding porins of the outer membrane
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, K؉
Immunodetection of EnvZ6His or 10His-OmpR proteins with polyclonal antibodies against EnvZ or the His tag was performed as described [34]. Influence of the His Tag of EnvZ on the Expression Pattern of ompF and ompC—To allow easy purification of EnvZ, six consecutive histidine residues were attached at the C-terminal end of the protein. The functionality of this protein was comparable with the untagged EnvZ because the ompC and ompF expression pattern was comparable with that of wild-type EnvZ as tested with E. coli strains WH56 and WH57, each transformed with plasmids, pFR29 or pFR29 – 6His, respectively
Summary
Materials—[␥-32P]ATP was purchased from Amersham Pharmacia Biotech. Goat anti-rabbit IgG-alkaline phosphatase conjugate was obtained from Biomol. Purification of EnvZ-6His—Proteins of everted membrane vesicles (140 mg) in buffer containing 50 mM Tris/HCl, pH 8.0, 10% glycerol, and 10 mM -mercaptoethanol were solubilized with 1% (w/v) n-decyl--Dmaltopyranoside (final protein concentration, 10 mg protein/ml). To obtain sufficient amounts of phosphorylated EnvZ in all experiments, the reaction was stopped after 2 min by the addition of an equal volume of 2ϫ concentrated SDS sample buffer [39]. Phosphorylation and Dephosphorylation Assays—Solubilized EnvZ or EnvZ in proteoliposomes (1 M) was incubated with 100 M [␥-32P]ATP (0.476 Ci/mmol) in phosphorylation buffer (50 mM Tris/HCl, pH 8.0, 10% glycerol, 2 mM dithiothreitol, and 110 M MgCl2) containing varying concentrations of test solutes (assay volume, 21 l). To test the complete signal transduction cascade in vitro, EnvZ in proteoliposomes (1 M), purified OmpR (4 M), C1-C2-C3 DNA fragment (5 M) [9] were mixed in phosphorylation buffer containing vari-
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