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.

SummaryThe 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 twocomponent 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.

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 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 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.In bacteria K ϩ plays an important role in the maintenance of intracellular pH, cell turgor and control of cellular enzyme activities (for a review, see reference 5), and, together with glutamate, it is also known to positively regulate the expression of certain osmoresponsive genes (23, 32). The intracellular K ϩ level is maintained by a variety of K ϩ uptake and efflux systems. The KdpFABC complex (K ϩ -dependent ATPase), encoded by the kdpFABC operon (1), is an inducible, high-affinity K ϩ transporter synthesized by Escherichia coli as an emergency system to scavenge K ϩ when other transporters cannot keep up with the cell's requirement for K ϩ (4). Expression of the kdpFABC operon is regulated by the products of the adjacent kdpDE genes (31). The membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE comprise a typical prokaryotic two-component signal transduction system (43). KdpD (894 amino acids; 99 kDa) consists of a cytoplasmic N-terminal domain and a cytoplasmic C-terminal domain interconnected by four transmembrane segments (46). Upon stimulus perception, KdpD undergoes autophosphorylation at His-673, and subsequently the phosphoryl group is transferred to Asp-52 of the response regulator KdpE (42). KdpE binds in its phosphorylated and dimerized form with high affinity at a 23-bp sequence immediately upstream of the canonical Ϫ35 and Ϫ10 regions of the kdpFABC promoter, thereby triggering kdpFABC transcription (41). The large input domain of the sensor kinase KdpD (about 660 ...