The extension of the fourth transmembrane helix of the sensor kinase KdpD of Escherichia coli is involved in sensing.
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.
Zimmann
Petra
P
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Barbarastr 11, D-49069, Osnabrück, Germany.
Steinbrügge
Anne
A
Schniederberend
Maren
M
Jung
Kirsten
K
Altendorf
Karlheinz
K
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
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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