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

Kirsten JungDagger and Karlheinz Altendorf

From the Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The kdpFABC operon, which encodes the structural genes for the high affinity K+ transport complex KdpFABC, is regulated by the sensor kinase KdpD and the response regulator KdpE. KdpD is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE. Here, we demonstrate that the phosphatase activity of KdpD is dependent on ATP, whereas GTP, ITP, CTP, ADP, and GDP have no effect. The phosphatase activity requires only ATP binding, because nonhydrolyzable analogs (adenosine-5'-[gamma -thio]triphosphate and adenosine-5'-[beta ,gamma -imido]triphosphate) work as well. However, KdpD proteins missing amino acids 12-128 are characterized by a phosphatase activity that is independent of ATP. These proteins are still able to respond to K+ starvation, but an increase in osmolarity is no longer sensed. Comparison of different KdpD sequences reveals a conserved motif in this amino acid region that is very similar to a classical ATP-binding site (Walker A motif). Replacement of the conserved Gly37, Lys38, and Thr39 residues in the consensus ATP-binding sequence results in a KdpD protein that causes a kdpFABC expression pattern comparable with that seen with KdpD proteins missing amino acids 12-128. However, in vitro phosphatase activity is comparable with that of wild-type KdpD. These results suggest that amino acids 12-128 of KdpD are important for its activity and that an additional ATP-binding site in the N-terminal region seems to be involved in modulation of the phosphatase activity.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

KdpD and KdpE, two proteins that regulate the expression of the kdpFABC operon in Escherichia coli (1), are members of the large family of sensor kinase/response regulator systems (see Refs. 2 and 3 for review). The corresponding genes are organized in the kdpDE operon, which is adjacent to the kdpFABC operon that encodes the structural genes of the high affinity K+ transport complex KdpFABC (4, 5). K+ is an important osmotic solute for the maintenance of turgor in bacterial cells (6), and the Kdp system can be characterized as an optional system to scavenge K+ from the environment. The stimulus that KdpD senses is believed to be a decrease in turgor pressure or some effect thereof (7).

Expression of kdpFABC is induced under K+-limiting growth conditions (below 2 mM). In mutants lacking all other K+-translocating transport systems (TrkG, TrkH, and Kup), kdpFABC is expressed in media containing 50 mM K+ or less. (7, 8). There is no correlation of kdpFABC expression with internal K+ concentration when this parameter is altered by changing medium osmolarity. Therefore, neither the external nor the internal concentration of K+ per se seems to be sensed but what might be called the "need" for K+ to maintain turgor. Control by turgor is supported by the finding that a sudden increase in medium osmolarity, which reduces turgor, is able to turn on expression of the kdpFABC operon transiently (7, 8). This model has been challenged by more recent findings (9, 10) demonstrating that there is a difference in expression of kdpFABC when the osmolarity of the medium is increased by a sugar or a salt. Analysis of mutant forms of KdpD that result in constitutive expression of kdpFABC independent of the K+ concentration of the medium but retain the ability to respond to changes in medium osmolarity led to the suggestion that KdpD senses two stimuli, decrease in turgor and K+ concentration (11).

The kdpD gene has been cloned, sequenced, and overexpressed (12). The gene product KdpD is an integral protein of the cytoplasmic membrane (13). Recently, KdpD was purified and reconstituted in an active form in proteoliposomes (14). This purified protein has autokinase activity. The phosphoryl group is subsequently transferred to the response regulator KdpE. Furthermore, KdpD catalyzes the dephosphorylation of purified KdpE~P (14).

According to hydropathy analysis of the primary amino acid sequence and the analysis of a series of KdpD-alkaline phosphatase (kdpD-phoA) and KdpD-beta -galactosidase (kdpD-lacZ) fusions as well as protease susceptibility experiments (15), the following secondary structure model was established (Fig. 1). KdpD consists of a large cytoplasmic N-terminal region, four putative transmembrane domains, and an extended cytoplasmic C-terminal region. Whereas the C-terminal domain shows high similarity to transmitter domains of other sensor kinases (16), the length of the N-terminal input domain is rather unusual.


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Fig. 1.   Schematic presentation of wild-type and different truncated forms of the sensor kinase KdpD. The model is based both on hydropathy plot analysis and studies with LacZ/PhoA fusions (15). The boxes represent the four transmembrane domains (TM1-TM4). Sequence motifs characteristic of transmitter domains of sensor kinases (H, N, G1, F and G2) are indicated in the upper part. In the lower part truncated KdpD proteins are schematically presented, where lines between the stippled bars comprise deleted parts of KdpD.

KdpD missing the four transmembrane domains became inactive in vivo and in vitro. However, proteins missing different parts of the soluble N-terminal domain could be phosphorylated in vitro, the phosphoryl group was transferred to KdpE, and they also contained phosphatase activity. The influence of the N-terminal truncations in KdpD on the transcriptional regulation of kdpFABC was tested in a trk- 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).

In this communication we describe for the first time the quantification of the rate of the phosphatase activity of KdpD. Furthermore, KdpD proteins missing amino acids 12-128 are characterized by a deregulated phosphatase activity. A conserved motif that is similar to a classical ATP-binding site (Walker A motif) can be found in this region. 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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [gamma -32P]ATP was purchased from Amersham Pharmacia Biotech. All nucleotides were purchased from Sigma. NAP 10 columns to remove ATP were obtained from Amersham Pharmacia Biotech. All other materials were reagent grade and obtained from commercial sources.

Bacterial Strains-- E. coli JM 109 [recA1 endA1 gyrA96 thi hsdR17 supE44 lambda -relA1 Delta (lac-proAB)/F' traD36 proA+B+ lacIqlacZDelta M15] (18) was used as carrier for the plasmids described. E. coli TKR2000 [Delta kdpFABCDE trkA405 trkD1 atp706] (19) harboring described plasmids was used for expression of kdpD from the tac promoter. E. coli HAK006 [Delta kdpABCD Delta (lac-pro) ara thi] carrying a kdpFABC promoter-lacZ fusion (20) was used to probe signal transduction in vivo.

Plasmids-- Plasmids pWP2-92 (Delta 12-228), pWP 4-92 (Delta 128-391), pWP6-92 (Delta 12-395) (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). All these plasmids are derivatives of pKK223-3, in which kdpD is under the control of the tac promoter. Furthermore, the kdpD gene was cloned into plasmid pBAD18 (22) using restriction sites SmaI and HindIII resulting in pBD. Subsequently, plasmids pWP2-92 (Delta 12-228), pWP4-92 (Delta 128-391), and pWP6-92 (Delta 12-395) were digested with PstI and StuI and ligated in similarly treated plasmid pBD. The resulting plasmids pBD (Delta 12-228), pBD (Delta 128-391), and pBD(Delta 12-395) were confirmed by restriction analysis.

Substitution of Gly37, Lys38, and Thr39 was achieved by PCR1 mutagenesis. The oligonucleotide primer was designed to change codons of Gly37, Lys38, and Thr39 to codons of Ala37, Ala38, and Cys39. The PCR product was purified in agarose gels and digested with appropriate restriction enzymes. The DNA fragment was isolated from agarose gels and ligated to similarly treated pPV5-1 resulting in pPV5-1, G37A,K38A,T39C. Subsequently, this kdpD construct was cloned into pBAD18 (22) using XmaI and HindIII restriction sites, resulting in plasmid pBD-G37A,K38A,T39C.

DNA Sequencing-- Mutations were verified by sequencing the length of the PCR-generated segment through the ligation junctions in double-stranded plasmid DNA, using the dideoxynucleotide termination method (23) and synthetic sequencing primers after alkaline denaturation of the DNA (24).

Preparation of Inverted Membrane Vesicles-- E. coli TKR2000 transformed with pPV5-1 or pPV5-1, G37A,K38A,T39C or pWP 2-92 (Delta 12-228), pWP 4-92 (Delta 128-391), and pWP6-92 (Delta 12-395) was grown aerobically at 37 °C in KML complex medium (1% tryptone, 0.5% yeast extract, and 1% KCl) supplemented with ampicillin (100 µg/ml). Cells were harvested at an absorbance at 600 nm of ~1.0. Inverted membrane vesicles were prepared as described previously (14).

Phosphorylation and Dephosphorylation Assays-- Inverted membrane vesicles containing KdpD (2 mg protein/ml) were incubated at room temperature in phosphorylation buffer, containing 50 mM Tris/HCl, pH 7.5, 10% glycerol, 0.5 M NaCl, 10 mM MgCl2, and 2 mM dithiothreitol. Phosphorylation was initiated by addition of 20 µM [gamma -32P]ATP (2.38 Ci/mmol). At different times, aliquots were removed and mixed with an equal volume of double concentrated SDS sample buffer (25). After incubation for 4.5 min, an equimolar amount of KdpE was added to the KdpD-containing fractions, and the incubation was continued. Further aliquots were removed at different times and mixed with SDS sample buffer as described above.

To test dephosphorylation, purified KdpE prepared as described (17) was phosphorylated in the following manner. Wild-type KdpD (4 mg/ml) was incubated in phosphorylation buffer, except that MgCl2 was replaced with 5 mM CaCl2, and phosphorylation was initiated with 20 µM [gamma -32P]ATP (2.38 Ci/mmol). After 5 min, purified KdpE (0.1 mg/ml) was added, and the incubation was continued for 1 min. KdpD-containing membrane vesicles were removed by centrifugation. ATP was removed by gel filtration through Sephadex G25 (preincubated in 50 mM Tris/HCl, pH 7.5, 10% glycerol, 2 mM dithiothreitol, 0.5 M NaCl). Purified KdpE~P was used immediately. Dephosphorylation was initiated by addition of 20 mM MgCl2, 20 µM ATP (or other nucleotides as specified), and inverted membrane vesicles containing KdpD (1 mg/ml). At different times aliquots were removed, and the reaction was stopped by addition of SDS sample buffer.

All samples were immediately subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (25). Shortly before stopping SDS-PAGE an [gamma -32P]ATP standard was loaded on the gels. Gels were dried, and phosphorylation of the proteins was detected by exposure of the gels to a phosphorscreen. Phosphorylated proteins were quantified by image analysis using the PhosphorImager system of Molecular Dynamics.

Probing Signal Transduction in Vivo-- In vivo signal transduction was probed with E. coli HAK006 transformed with the plasmids described. Cells were grown in TY medium (1% tryptone, 0.5% yeast extract) (11) or minimal medium (26) supplemented with NaCl and KCl as indicated. Cells were grown to midlogarithmic growth phase and harvested by centrifugation. beta -Galactosidase activity was determined as described (27) and is given in Miller units.

Analytical Procedures-- Protein was assayed by the method of Peterson (28) with bovine serum albumin as standard. Proteins were separated by SDS-PAGE (25) using 9 or 12% acrylamide gels. Immunodetection of KdpD proteins with polyclonal antibodies against KdpD was performed according to (13).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Kinase and Phosphotransfer Activities and the Influence of N-terminal Truncations in KdpD-- Inverted membrane vesicles containing equal amounts of either wild-type or truncated KdpD, as judged by Western blot analysis (data not shown), were tested for kinase activity. The time courses of autophosphorylation of different truncated KdpD proteins in comparison with wild-type KdpD is shown in Fig. 2. Autophosphorylation was tested to be linear within 0.5 min. Furthermore, transfer of the phosphoryl group to KdpE was determined. The transfer of the phosphoryl group to KdpE was very fast (within 15 s the phosphotransfer was complete). Phosphotransfer was detectable for all truncated KdpD proteins (data not shown).


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Fig. 2.   Autophosphorylation of wild-type and truncated forms of KdpD with [gamma -32P]ATP. Time-dependent phosphorylation of inverted membrane vesicles was carried out as described under "Experimental Procedures." Proteins were separated by SDS-PAGE. The amount of KdpD~P was quantified with a PhosphorImager using [gamma -32P]ATP as standard. bullet , wild-type KdpD; black-diamond , KdpD (Delta 128-391); square , KdpD (Delta 12-228); triangle , KdpD (Delta 12-395).

Phosphatase Activity of KdpD and the Influence of N-terminal Truncations-- In addition to the kinase activity KdpD catalyzes also the dephosphorylation of phosphorylated KdpE (14). KdpE~P itself is very stable; within 2 h no major loss of the phosphoryl group was detected. Dephosphorylation of KdpE~P was initiated by the addition of inverted membrane vesicles containing KdpD, 20 mM MgCl2, and 20 µM ATP. Within about 5 min half of the amount of KdpE~P was dephosphorylated (Fig. 3A). The rate of dephosphorylation of wild-type KdpD was comparable with that of the truncated forms. For wild-type KdpD only ATP was effective to stimulate the phosphatase activity; GTP, ITP, CTP, ADP, and GDP were without effect. Furthermore, nonhydrolyzable ATP analogs, ATP-gamma -S and AMP-PNP, were as effective as ATP (data not shown). In the absence of ATP a very slow dephosphorylation was observed when wild-type KdpD and KdpD (Delta 128-391) were tested (Fig. 3B). Interestingly, KdpD (Delta 12-228) and KdpD (Delta 12-395) are characterized by a phosphatase activity that is independent of the presence of ATP.


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Fig. 3.   Dephosphorylation of KdpE~P. KdpE was phosphorylated as described under "Experimental Procedures." Dephosphorylation was initiated by addition of wild-type KdpD (bullet ), KdpD (Delta 128-391) (black-diamond ), KdpD (Delta 12-228) (square ), or KdpD (Delta 12-395) (triangle ) in inverted membrane vesicles in the presence (A) or absence (B) of 20 µM ATP. As a control inverted membrane vesicles without KdpD were added (open circle ). Proteins were separated by SDS-PAGE. The amount of phosphorylated KdpE was determined with a PhosphorImager using [gamma -32P]ATP as standard.

Influence of N-terminal Truncations in KdpD on the Regulation of kdpFABC Expression-- It has been found previously that in wild-type strains kdpFABC is expressed when medium concentration of K+ is below 2 mM. Furthermore, an increase in medium osmolarity at constant K+ concentration, a maneuver that reduces turgor, caused expression of kdpFABC (7). Signal transduction mediated by truncated forms of KdpD was previously tested in E. coli deleted for other K+ uptake systems (TrkG and TrkH) leaving Kdp as the main K+ transport system. Because of the importance of the Kdp system as the major K+ 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+ uptake systems, TrkH, TrkG, and Kup. This strain lacks the functional kdpFABC operon as well as the kdpD gene on the chromosome but contains the intact kdpE gene under the control of its own promoter. In addition, this strain harbors a kdpFABC promoter-lacZ fusion gene on the chromosome. Because the amount of regulatory proteins is very critical in signal transduction (high levels of KdpD prevent complementation of a kdpD null strain),2 E. coli HAK006 was transformed with plasmids pBD and its derivatives. In plasmid pBD kdpD is under the control of the arabinose promoter (22). When cells were grown in the absence of an inducer (arabinose) and in the presence of the repressor glucose, the amount of KdpD produced was sufficient to complement a kdpD null strain. The truncated proteins were tested for their response to an increase in osmolarity and K+ limitation in comparison with wild-type KdpD (Table I). In response to an increase of osmolarity beta -galactosidase activities of cells producing wild-type KdpD or KdpD (Delta  128-391) increased, whereas only basal levels of beta -galactosidase activity were detectable in cells producing KdpD (Delta 12-228) or KdpD (Delta 12-395). Furthermore, K+ limitation resulted in high beta -galactosidase activities when cells produced wild-type KdpD or KdpD (Delta 128-391). In the case of KdpD (Delta 12-228) and KdpD (Delta 12-395), beta -galactosidase was detectable when cells were cultivated at very low K+ concentrations. However, beta -galactosidase activity was much lower in comparison with that of wild-type KdpD.

                              
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Table I
Influence of alterations in the N-terminal domain in KdpD on the regulation of kdpFABC expression by K+ concentration and osmotic stress
E. coli HAK006 was transformed with plasmid pBD, its derivatives carrying the indicated deletions in kdpD, or pBD-G37A,K38A,T39C. Cells were grown to midlogarithmic growth phase in minimal medium containing different amounts of K+ or in TY medium in the presence of different NaCl concentrations (the basal concentration of K+ in this medium was determined to be 6 mM by flame photometry). beta -Galactosidase activities were determined as described under "Experimental Procedures" and is given in Miller units (27). The data presented represent average values obtained in at least three independent experiments.

Characterization of KdpD-G37A,K38A,T39C-- Sequence comparison of the first 200 amino acids of so far six known KdpD sequences of different microorganisms revealed that a conserved motif exists that is similar to a classical ATP-binding site (Walker A motif) (29) (Fig. 4). In addition, we found that KdpD proteins missing these amino acids (KdpD(Delta 12-395) and KdpD (Delta 12-228)) are characterized by a phosphatase activity that is independent of ATP and cause deregulation of kdpFABC expression. To test whether KdpD contains a second ATP-binding site, site-directed mutagenesis was used to inactivate this site. Therefore, codons for Gly37, Lys38, and Thr39 were replaced by codons for Ala37, Ala38, and Cys39 in kdpD. The resulting KdpD protein was tested for kinase and phosphatase activity. The rate of autophosphorylation was comparable with that of wild-type KdpD; after addition of KdpE the phosphoryl group was rapidly transferred (data not shown). KdpD-G37A,K38A,T39C catalyzed the dephosphorylation of KdpE~P. In the absence of ATP the rate of dephosphorylation was very slow, and it could be stimulated in the presence of ATP. Rates were comparable with that of wild-type KdpD or KdpD(Delta 128-391) (data not shown). Furthermore, transcriptional induction of kdpFABC was tested using E. coli strain HAK006 transformed with plasmid pBD-G37A,K38A,T39C. Inactivation of the proposed ATP-binding site impaired kdpFABC expression in response to an increase of the osmolarity of the medium (Table I). As shown before with truncated proteins missing amino acids 12-395 or 12-228, respectively, KdpD-G37A,K38A,T39C showed a modest ability to induce reporter gene expression in response to K+ starvation (Table I).


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Fig. 4.   Homology among the first 200 amino acids of KdpD proteins from E. coli (E.c.), Clostridium acetobutylicum (C.a.), Mycobacterium tuberculosis (M.t.), Rathayibacter rathayi (R.r.), Synechocystis spec. (S.s.), and Streptomyces coelicolor (S.c.) (EMBL data bank). Identical regions are shaded dark, and regions displaying high homology are shaded light. Areas with internal gaps are indicated by a dash. Marked with boxes are two motifs that are very similar to the Walker A and Walker B motif (40). The sequence of a classical Walker A motif is shown above (29). The figure was created with the PIMA Multiple Sequence Alignment program of the BCM Search Launcher (Human Genome Center, Baylor College of Medicine, Houston, TX).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Dephosphorylation of the response regulator is well studied in various sensor kinase/response regulator systems and characterized by diverse mechanisms (16). The sensor kinase EnvZ by itself is able to enhance the rate of dephosphorylation of phospho-OmpR (31-33). In the case of NRII an auxiliary protein PII is involved (34-36). In the chemotaxis system, dephosphorylation of CheY is mediated by a separate protein, CheZ (37), and in the process of sporulation of Bacillus subtilis different phosphatases have been identified (38).

Using purified and reconstituted KdpD we have recently shown that dephosphorylation of KdpE~P is only dependent on the sensor kinase (14). In this communication we demonstrate for the first time that the rate of dephosphorylation can be significantly increased in the presence of ATP or nonhydrolyzable ATP analogs. In contrast, other nucleotides do not have an effect at all. The dephosphorylation activity of EnvZ is also stimulated by ATP, but in contrast to KdpD, also by ADP (31-33).

In comparison with other sensor kinases, KdpD contains an extended hydrophilic N-terminal domain of 400 amino acids exposed to the cytoplasm. This N-terminal domain is characterized by a high degree of homology between the so far known KdpD sequences from different organisms (Fig. 4). It is tempting to speculate that this domain, in addition to the four transmembrane helices, is involved in stimulus perception.

We found that the stimulation of phosphatase activity of truncated forms of KdpD missing amino acid 12-128 is no longer dependent on ATP. Furthermore, we demonstrated that amino acids 12-128 of the N-terminal domain of KdpD are important for the regulation of kdpFABC expression. KdpD(Delta 12-395) and KdpD(Delta 12-228) are not able to respond to an increase in osmolarity raised by the addition of NaCl. When cells synthesizing these truncated proteins were grown under K+-limiting conditions, the kdpFABC operon was expressed, but the level of expression was much lower in comparison with that of wild-type KdpD. In contrast, the truncation of amino acids 128-391 results in a KdpD protein that gave rise to the same expression level of the kdpFABC operon as wild-type KdpD.

In addition to the strong dependence of the phosphatase activity on the presence of ATP we found that amino acids 31-39 comprise a motif that is conserved among the KdpD sequences (Fig. 4) and that is very similar to a classical ATP binding site (Walker A motif) of many ATP-requiring enzymes (29). As indicated in Fig. 4, a Walker B motif can also be found. The most frequently published form of the Walker A motif is GXXXXGKT (39, 40). The motif found in KdpD has one more X between the first and second Gs. There is one other ATP-binding site found in the dethiobiotin synthetase that has the same insertion. Structural analysis of this protein reveals that the dehydral angles of the conserved residues of the motif are identical to those of other proteins containing the classical motif (41). Therefore, we proposed that KdpD contains a second, regulatory ATP-binding site. To test this hypothesis, we replaced together the conserved residues Gly37, Lys38, and Thr39 by site-directed mutagenesis. In this case kdpFABC expression is reduced to the same extent as it is seen with KdpD proteins missing this putative ATP-binding site. This results are in favor of the existence of a regulatory ATP-binding site in the N-terminal domain of KdpD.

It is conceivable that the low level of kdpFABC expression is because of a decrease of the amount of KdpE~P. Because in vitro no differences were seen in the kinase activities of KdpD proteins missing this site, it is suggested that ATP-binding regulates the phosphatase activity of KdpD. Indeed, we observed a deregulation of the phosphatase activity of KdpD proteins missing amino acids 12-128. However, the deregulation of the phosphatase activity in vitro was not observed with KdpD-G37A,K38A,T39C. Therefore, an interaction of the N- and the C-terminal domain may also contribute to the regulation of the phosphatase activity. An interaction of the input and transmitter domain depending on the stimulus might influence the switch between kinase and phosphatase activity in such a way that through conformational changes the accessibility of the putative regulatory ATP-binding site may vary and thereby the phosphatase activity might be tightly regulated. Although it is known that upon an osmotic upshift intracellular ATP concentration varies (42), it is too premature to draw conclusions about the interplay between ATP concentration and activity of KdpD.

There are to our knowledge only two other sensor kinases known that contain an additional nucleotide binding site (Walker A motif). In ChvG from Agrobacterium tumefaciens this putative site is located in the transmitter domain (43). The unorthodox sensor kinase BvgS from Bordetella pertussis contains such a motif in its linker region. Individual replacements of amino acid residues of this motif caused the inactivation of BvgS indicating the functional importance of this site (30).

In summary, we could show here that the phosphatase activity of KdpD can be increased in the presence of ATP or ATP analogs. Furthermore, KdpD proteins missing amino acids 12-128 are characterized by a deregulated phosphatase activity in vitro. Finally, KdpD proteins missing a conserved motif in the N-terminal domain, which is similar to a classical ATP-binding site (Walker A), either by truncation or mutagenesis diminish kdpFABC expression in response to K+ limitation or osmotic upshock.

    ACKNOWLEDGEMENTS

We thank Mechthild Krabusch and Britta Brickwedde for excellent technical assistance and Markus Veen for providing data for Table I. We also thank M. Lucassen for providing purified response regulator KdpE, Dr. T. Mizuno for E. coli strain HAK006, and Dr. J. Beckwith for plasmid pBAD18.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 49-541-969-2276; Fax: 49-541-969-2870; E-mail: jung_k{at}sfbbio1.biologie.Uni-Osnabrueck.DE.

1 The abbreviations used are: PCR, polymerase chain reaction; ATP-gamma -S, adenosine-5'-[gamma -thio]triphosphate; AMP-PNP, adenosine-5'-[beta , gamma -imido]triphosphate; PAGE, polyacrylamide gel electrophoresis.

2 K. Jung, unpublished information.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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