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; Ludwig-Maximilians-Universität München, Department Biologie I, Bereich Mikrobiologie, Maria-Ward-Str. 1a, D-80638 München

^* To whom correspondence should be addressed. Email: altendorf{at}biologie.uni-osnabrueck.de.

	Abstract

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 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 in turn, also the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K⁺, both activities were not affected 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 K⁺limitation and osmotic upshock, both of which may not be separated mechanistically.