Absolute quantification of the Kdp subunits of Escherichia coli by multiple reaction monitoring.

The sensor kinase/response regulator system KdpD/KdpE of Escherichia coli regulates the expression of the kdpFABC operon, encoding the high-affinity KdpFABC potassium (K(+) )-transport complex. Additionally, it has been suggested that the kdpDE operon itself is subjected to autoregulation by its gene products KdpD and KdpE. However, since kdpFABC and kdpDE expression has mainly been studied on the transcriptional level, accurate information on absolute amounts and the stoichiometric subunit composition of KdpFABC and KdpD/KdpE under K(+) -limiting and K(+) -nonlimiting growth conditions are lacking. In this study, we used highly sensitive mass spectrometric methods to quantify the amount of subunits of the Kdp(F)ABC complex and KdpD/KdpE. Data-dependent shotgun MS was used to assess protein coverage and accessible peptides. Absolute amounts of Kdp(F)ABC and KdpD/KdpE were quantified by targeted MRM analysis in the presence of corresponding heavy labeled standard peptides. Baseline synthesis of Kdp(F)ABC and KdpD/KdpE was found to be in the attomolar range under K(+) -nonlimiting conditions. Under K(+) -limitation, synthesis of Kdp(F)ABC (KdpA:KdpB:KdpC ratio of 1:1:1) was amplified more than 100-fold, whereas only a tenfold amplification of KdpD/KdpE (KdpD:KdpE ratio of 1:4) was observed. The results obtained provide a solid basis for follow-up studies on the dynamic regulation of the Kdp system.

The sensor kinase KdpD of Escherichia coli senses external K+.

The Kdp system of Escherichia coli is composed of the high-affinity K(+) transporter KdpFABC and the two regulatory proteins KdpD (sensor kinase) and KdpE (response regulator), which constitute a typical two-component system. The kdpFABC operon is induced under K(+) -limiting conditions and, to a lesser extent, under high osmolality in the medium. In search for the stimulus sensed by KdpD, we studied the inhibitory effect of extracellular K(+) on the Kdp system at pH 6.0, which is masked by unspecific K(+) transport at higher pH values. Based on KdpD derivatives carrying single aspartate replacements in the periplasmic loops which are part of the input domain, we concluded that the inhibition of the Kdp system at extracellular K(+) concentrations above 5 mM is mediated via KdpD/KdpE and not due to inhibition of the K(+) -transporting KdpFABC complex. Furthermore, time-course analyses of kdpFABC expression revealed that a decline in the extracellular K(+) concentration efficiently stimulates KdpD/KdpE-mediated signal transduction. In this report we provide evidence that the extracellular K(+) concentration serves as one of the stimuli sensed by KdpD.

Influence of K+-dependent membrane lipid composition on the expression of the kdpFABC operon in Escherichia coli.

The membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE regulate the expression of the kdpFABC operon coding for the high affinity potassium uptake system KdpFABC in Escherichia coli. The signal transduction cascade of this two component system is activated under K(+)-limiting conditions in the medium, but is less sensitive to high osmolality. In order to test whether K(+) limitation affects membrane phospholipid composition and whether this change affects kdpFABC expression, we analysed the phospholipid composition of E. coli under these conditions. Our measurements revealed that there is an increase in the cardiolipin (CL) content during the exponential growth phase at the expense of the zwitterionic phospholipid phosphatidylethanolamine. The higher anionic phospholipid content occurs along with an increase of transcriptional activity of the cls gene coding for CL synthase. Furthermore, in vivo studies with E. coli derivatives carrying mutations in genes coding for enzymes involved in phospholipid biosynthesis revealed that the increase in the anionic lipid composition enhances the expression rate of the kdpFABC operon. Finally, we show that kinase activity of KdpD is stimulated in its native membrane environment by fusion with liposomes of anionic, but reduced with liposomes of zwitterionic phospholipids.

Reduction of turgor is not the stimulus for the sensor kinase KdpD of Escherichia coli.

Stimulus perception by the KdpD/KdpE two-component system of Escherichia coli is still controversial with respect to the nature of the stimulus that is perceived by the sensor kinase KdpD. Limiting potassium concentrations in the medium or high osmolality leads to KdpD/KdpE signal transduction, resulting in kdpFABC expression. It has been hypothesized that changes in turgor are sensed by KdpD through alterations in the physical state of the cytoplasmic membrane. However, in this study the quantitative determination of expression levels of the kdpFABC operon revealed that the system responds very effectively to K(+)-limiting conditions in the medium but barely and to various degrees to salt and sugar stress. Since the current view of stimulus perception calls for mainly intracellular parameters, which might be sensed by KdpD, we set out to test the cytoplasmic concentrations of ATP, K(+), Na(+), glutamate, proline, glycine, trehalose, putrescine, and spermidine under K(+)-limiting conditions. As a first result, the determination of the cytoplasmic volume, which is a prerequisite for such measurements, revealed that a transient shrinkage of the cytoplasmic volume, which is indicative of a reduction in turgor, occurred only under osmotic upshift but not under K(+)-limiting conditions. Furthermore, the intracellular ATP concentration significantly increased under osmotic upshift, whereas only a slight increase occurred after a potassium downshift. Finally, the cytoplasmic K(+) concentration rose severalfold only after an osmotic upshock. For the first time, these data indicate that stimulus perception by KdpD correlates neither with changes in the cytoplasmic volume nor with changes in the intracellular ATP or K(+) concentration or those of the other solutes tested. In conclusion, we propose that a reduction in turgor cannot be the stimulus for KdpD.

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.

An atypical KdpD homologue from the cyanobacterium Anabaena sp. strain L-31: cloning, in vivo expression, and interaction with Escherichia coli KdpD-CTD.

The kdpFABC operon of Escherichia coli, coding for the high-affinity K(+) transport system KdpFABC, is transcriptionally regulated by the products of the adjacently located kdpDE genes. The KdpD protein is a membrane-bound sensor kinase consisting of a large N-terminal domain and a C-terminal transmitter domain interconnected by four transmembrane segments (the transmembrane segments together with the C-terminal transmitter domain of KdpD are referred to as CTD), while KdpE is a cytosolic response regulator. We have cloned and sequenced the kdp operon from a nitrogen-fixing, filamentous cyanobacterium, Anabaena sp. strain L-31 (GenBank accession. number AF213466). The kdpABC genes are similar in size to those of E. coli, but the kdpD gene is short (coding only for 365 amino acids), showing homology only to the N-terminal domain of E. coli KdpD. A kdpE-like gene is absent in the vicinity of this operon. Anabaena KdpD with six C-terminal histidines was overproduced in E. coli and purified by Ni(2+)-nitrilotriacetic acid affinity chromatography. With antisera raised against the purified Anabaena KdpD, the protein was detected in Anabaena sp. strain L-31 membranes. The membrane-associated or soluble form of the Anabaena KdpD(6His) could be photoaffinity labeled with the ATP analog 8-azido-ATP, indicating the presence of an ATP binding site. The coproduction of Anabaena KdpD with E. coli KdpD-CTD decreased E. coli kdpFABC expression in response to K(+) limitation in vivo relative to the wild-type KdpD-CTD protein. In vitro experiments revealed that the kinase activity of the E. coli KdpD-CTD was unaffected, but its phosphatase activity increased in the presence of Anabaena KdpD(6His). To our knowledge this is the first report where a heterologous N-terminal domain (Anabaena KdpD) is shown to affect in trans KdpD-CTD (E. coli) activity, which is just opposite to that observed for the KdpD-N-terminal domain of E. coli.