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The Extension of the Fourth Transmembrane Helix of the Sensor Kinase KdpD of Escherichia coli Is Involved in Sensing

Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany,¹ Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Strasse 1a, D-80638 München, Germany²

Received 20 June 2007/ Accepted 7 August 2007

The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K⁺ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K⁺ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K⁺ concentration. However, 3- to 14-fold induction was observed under extreme K⁺-limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K⁺, both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K⁺ limitation and osmotic upshift, which may not be separated mechanistically.

Published ahead of print on 17 August 2007.

This article has been cited by other articles:

• Hamann, K., Zimmann, P., Altendorf, K. (2008). Reduction of Turgor Is Not the Stimulus for the Sensor Kinase KdpD of Escherichia coli. J. Bacteriol. 190: 2360-2367 [Abstract] [Full Text]