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. 2006 Mar;188(5):1950-8.
doi: 10.1128/JB.188.5.1950-1958.2006.

The cytoplasmic C-terminal domain of the Escherichia coli KdpD protein functions as a K+ sensor

Marina C Rothenbücher  1 , Sandra J Facey, Dorothee Kiefer, Marina Kossmann, Andreas Kuhn
Affiliations

The cytoplasmic C-terminal domain of the Escherichia coli KdpD protein functions as a K+ sensor

Marina C Rothenbücher et al. J Bacteriol. 2006 Mar.

Abstract

The KdpD protein is a K(+) sensor kinase located in the cytoplasmic membrane of Escherichia coli. It contains four transmembrane stretches and two short periplasmic loops of 4 and 10 amino acid residues, respectively. To determine which part of KdpD functions as a K(+) sensor, genetic variants were constructed with truncations or altered arrangements of the transmembrane segments. All KdpD constructs were tested by complementation of an E. coli kdpD deletion strain for their ability to grow at a K(+) concentration of 0.1 mM in the medium. A soluble protein composed of the C-terminal cytoplasmic domain was able to complement the kdpD deletion strain. In addition, analysis of the beta-galactosidase activity of an E. coli strain which carries a transcriptional fusion of the upstream region of the kdpFABC operon and a promoterless lacZ gene revealed that this soluble KdpD mutant responds to changes in the K(+) concentration in the extracellular medium. The results suggest that the sensing and response functions are both located in the C-terminal domain and might be modulated by the N-terminal domain as well as by membrane anchoring.

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Figures

FIG. 1.
FIG. 1.
Schematic representation of KdpD and variants showing the predicted membrane topologies of the proteins used in this study. The numbered boxes represent the transmembrane domains. KdpD (wild type), KdpD-N, KdpD-C, N1-2C, N3-4C, and N1-4C are shown with their associated cytoplasmic regions. H4+C contains transmembrane helix 4 and the C-terminal cytoplasmic region, and H4+C+RR contains two arginine residues introduced N-terminally to helix 4. The C494-894 and C499-894 proteins have a truncation or complete deletion of helix 4, respectively.
FIG. 2.
FIG. 2.
KdpD mutants with transmembrane segments are found in the membrane fraction. Plasmids carrying the different engineered kdpD genes were transformed into E. coli MC1061. Cultures were grown in LB medium to an OD600 of 0.2 and then induced with 0.2% arabinose for 3 h. The supernatant (S) and pellet (P) fractions after sodium carbonate extraction were prepared as described in Materials and Methods.
FIG. 3.
FIG. 3.
The cytoplasmic C-terminal domain is sufficient to sense changes in external K+ concentration. Strain HAK006 was transformed with plasmids encoding the various KdpD variants. Cells were grown to mid-logarithmic phase in minimal medium containing 0.1 mM KCl or 6 mM KCl. The β-galactosidase activity was determined as described in Materials and Methods and is given in Miller units (17). The data represent the means of triplicate determinations.
FIG. 4.
FIG. 4.
Activation of KdpD, KdpD-C, and C499-894 is specific for K+. HAK006 cells producing KdpD (A), KdpD-C (B), or C499-894 (C) were grown to mid-logarithmic phase in minimal medium containing 6 mM KCl. Cells were washed with minimal medium containing 0.1 mM KCl and transferred into fresh medium containing 0.1 mM KCl. Then, 6 mM RbCl or 6 mM LiCl was added. A control culture was resuspended in medium containing 6 mM KCl. The β-galactosidase activity was determined at the indicated time points and normalized to the maximum β-galactosidase activity (100% of control Miller units) of the KdpD+ culture growing in medium containing 0.1 mM KCl.
FIG. 5.
FIG. 5.
KdpD-C and C499-894 have inactivation kinetics like KdpD. (A) HAK006 cells expressing KdpD, KdpD-C, or C499-894 were grown to mid-logarithmic phase in minimal medium containing 0.1 mM KCl. Samples were taken at the indicated time points after addition of 6 mM KCl and assayed for β-galactosidase activity. (B) HAK006 cells expressing KdpD, KdpD-C, or C499-894 were grown to mid-logarithmic phase in minimal medium containing 0.1 mM KCl and divided into aliquots. The indicated amounts of KCl were added to the cultures, and growth was continued for 6 h. The β-galactosidase activity was determined and normalized to the value of the initial culture for each sample.
FIG. 6.
FIG. 6.
Activation (A to C) and inactivation kinetics (D) of KdpD, KdpD-C, and C499-894 in HMK006. HMK006 cells expressing KdpD (A), KdpD-C (B), or C499-894 (C) were grown overnight in minimal medium containing 10 mM KCl. Cells were washed with minimal medium containing 0.1 mM KCl and diluted 1:100 into fresh minimal medium containing 0.1 mM KCl. A control culture was resuspended in medium containing 10 mM KCl. The β-galactosidase activity was determined at the indicated time points and normalized to the maximum β-galactosidase activity (100% of control Miller units) of the KdpD+ culture growing in medium containing 0.1 mM KCl. (D) HMK006 cells expressing KdpD, KdpD-C, or C499-894 were grown in minimal medium containing 0.1 mM KCl to mid-logarithmic phase. Then, 10 mM KCl was added. Samples were taken at the indicated time points after addition of 10 mM KCl and assayed for β-galactosidase activity.
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