Characterization of Truncated Forms of the KdpD Protein, the Sensor Kinase of the K+-translocating Kdp System of Escherichia coli

Abstract

The expression of the kdpFABC operon, coding for the K+-translocating Kdp system, is controlled by the two regulatory proteins, KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems. This study describes the construction and analysis of KdpD sensor kinases, in which different deletions in the N-terminal part of the protein were introduced. Truncated KdpD proteins, in which the membrane-spanning segments were deleted, had lost their phosphorylation capacity. Truncated KdpD proteins, in which the four membrane-spanning helices were untouched, were still phosphorylated, and the phosphoryl group could be transferred to the response regulator KdpE in vitro. Furthermore, these truncated KdpD proteins cause dephosphorylation of KdpE(P), which is comparable with that of the wild-type protein. To investigate the effect of the deletions on signal transduction in vivo the corresponding kdp genes were transferred to the chromosome. Growth studies with the mutant strains are in accord with the data obtained from the in vitro studies. Furthermore, kdp expression was investigated using a KdpA-LacZ fusion. The data obtained support the notion that the extent of kdp expression is modulated by the N-terminal part of KdpD.

The Hydrophilic N-terminal Domain Complements the Membrane-anchored C-terminal Domain of the Sensor Kinase KdpD ofEscherichia coli

“…This presumption is supported by the differentiated response of KdpD proteins with individual substitutions of Arg residues toward K ϩ limitation and osmotic upshock (9). Compared with other known sensor kinases, KdpD is the only protein that has a large, hydrophilic N-terminal domain of about 400 amino acids in length (17,18) (Fig. 1) that this domain is more conserved then other regions of KdpD.…”

Truncation of Amino Acids 12–128 Causes Deregulation of the Phosphatase Activity of the Sensor Kinase KdpD of Escherichia coli

“…Signal transduction mediated by truncated forms of KdpD was previously tested in E. coli deleted for other K ϩ uptake systems (TrkG and TrkH) leaving Kdp as the main K ϩ transport system. Because of the importance of the Kdp system as the major K ϩ uptake system, kdpFABC expression was never completely blocked (17). Therefore, we tested the transcriptional induction of kdpFABC with E. coli HAK006 (11) that synthesizes the constitutive K ϩ uptake systems, TrkH, TrkG, and Kup.…”

“…The influence of the N-terminal truncations in KdpD on the transcriptional regulation of kdpFABC was tested in a trk Ϫ background. It was shown that the truncation of amino acids 12-128 influences the level of kdpFABC expression, whereas the truncation of amino acids 128 -391 exhibits expression levels that were comparable with wild-type KdpD (17).…”

“…The strong dependence of the phosphatase activity of KdpD on ATP and alteration of the proposed nucleotide binding site by site-directed mutagenesis provide first evidence that ATP-binding modulates KdpD activity. (17), and pPV5-1 (21) were used. Plasmid pPV5-1 is a derivative of plasmid pPV5 (12), which contains five unique restriction sites that were introduced by silent mutation (21).…”

The N-terminal Input Domain of the Sensor Kinase KdpD of Escherichia coli Stabilizes the Interaction between the Cognate Response Regulator KdpE and the Corresponding DNA-binding Site

“…Before use, 10His-KdpE was dialyzed against buffer E without KCl and imidazole. Alternatively, KdpE was purified following a method described before (12).…”