Browsing by Subject "Sensorkinase"

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Funktionelle Untersuchung der Sensorkinase KdpD von Escherichia coli mit Hilfe verschiedener KdpD-Deletionsmutanten
(2007) Rothenbücher, Marina; Kuhn, Andreas
The high affinity K+ transport system KdpFABC is one of several uptake systems that accumulate K+ in Escherichia coli. Expression of the kdpFABC operon is under control of the regulatory proteins KdpD and KdpE, which constitute a typical sensor kinase/response regulator system. KdpD is an integral protein of the cytoplasmic membrane. The N-terminal domain, the 4 helices and 200 amino acids of the cytoplasmic C-terminal domain are accredited to be involved in the signal input function. Surprisingly, a mutant (KdpD-C) lacking the N-terminal domain, helix 1 and 2 is a functional K+ sensor, which is able to detect the changes in K+ concentration in the medium. To investigate which parts of the KdpD protein are essential for signal transduction, various truncated KdpD variants were constructed and analyzed. The results show that the fragment C499-894, which contains only the cytoplasmic C-terminal domain of KdpD, is able to recognise the increase in K+ concentration in medium and reduce the level of activity. This mini sensor is also able to discriminate between Li+, Rb+ and K+ ions like the wild-type KdpD. A plasmid coding for this mini sensor allows a kdpD deletion strain to grow under K+-limited conditions in medium. Presumably, the signal perceived by KdpD is in the periplasm, but how is the signal transmitted to the cytoplasmic domain of KdpD that controls activity? Perhaps KdpD is not the direct sensor, an additional component in the membrane might sense the signal and communicate with KdpD. Further research with sodium carbonate extraction to determine the membrane localisation of C499-894 showed the protein mainly found in the supernatant, suggesting C499-894 is a soluble protein, although C499-894 could be attached to the membrane to come in contact with an unknown membrane protein. Looking for the unknown component two protein candidates were found by co-purification, the cytochrome oxidase A (CyoA) and the glucosamine-6-phosphate synthase (GlmS). To find an interaction between these proteins and KdpD more research has to be done.