title: The extension of the fourth transmembrane helix of the sensor kinase KdpD of Escherichia coli is involved in sensing.
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 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.
authors: Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Barbarastr 11, D-49069, Osnabrück, Germany.
authors: Petra
authors: P
authors: Zimmann
authors: Anne
authors: A
authors: Steinbrügge
authors: Maren
authors: M
authors: Schniederberend
authors: Kirsten
authors: K
authors: Jung
authors: Karlheinz
authors: K
authors: Altendorf
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D027682:Cation Transport Proteins / Q000096:biosynthesis*; D004252:DNA Mutational Analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology*; D016296:Mutagenesis; D009997:Osmotic Pressure; D011188:Potassium / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D013395:Sucrose / Q000378:metabolism
chemical_list: D027682:Cation Transport Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D013395:Sucrose; C489580:KdpD protein, E coli; D011494:Protein Kinases; D011188:Potassium
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