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• W2074001058 abstract "In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase ofEscherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute. The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His6 protein was functional in vitro andin vivo. KdpD-His6 was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind 2-azido-ATP. KdpD-His6 was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His6exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE. Furthermore, KdpD-His6 was found to be the only protein that mediates dephosphorylation of KdpE∼P. In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase ofEscherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute. The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His6 protein was functional in vitro andin vivo. KdpD-His6 was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind 2-azido-ATP. KdpD-His6 was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His6exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE. Furthermore, KdpD-His6 was found to be the only protein that mediates dephosphorylation of KdpE∼P. Kdp is one of several transport systems that accumulate K+ in bacteria. Among these systems, Kdp has the highest affinity for K+, and it is only expressed when other systems are unable to meet the cells' need for K+. Expression of the kdpFABC operon, coding for the K+-translocating Kdp ATPase, is regulated by two proteins (KdpD and KdpE) that belong to the class of sensor kinase/response regulator systems (see Refs. 1Altendorf K. Epstein W. Lee A.G. Biomembranes.5. Jai Press, Inc., London, England1996: 403-420Google Scholar and 2Altendorf K. Voelkner P. Puppe W. Res. Microbiol. 1994; 145: 374-381Crossref PubMed Scopus (30) Google Scholar for review). The kdpDgene has been cloned, sequenced, and overexpressed (3Walderhaug M.O. Polarek J.W. Voelkner P. Daniel J.M. Hesse J.E. Altendorf K. Epstein W. J. Bacteriol. 1992; 174: 2152-2159Crossref PubMed Google Scholar). The gene product KdpD is a membrane-bound protein located in the cytoplasmic membrane (3Walderhaug M.O. Polarek J.W. Voelkner P. Daniel J.M. Hesse J.E. Altendorf K. Epstein W. J. Bacteriol. 1992; 174: 2152-2159Crossref PubMed Google Scholar, 4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). According to hydropathy analysis of the primary amino acid sequence, KdpD consists of an extended cytoplasmic N-terminal region, four putative transmembrane domains, and an extended cytoplasmic C-terminal region (see Fig. 1). This model is strongly supported by the analysis of a series of KdpD-alkaline phosphatase (kdpD-phoA) and KdpD-β-galactosidase (kdpD-lacZ) fusions as well as protease susceptibility studies (5Zimmann P. Puppe W. Altendorf K. J. Biol. Chem. 1995; 270: 28282-28288Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Phosphorylation of KdpD is readily demonstrated in vitro by crude cell extracts (4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar, 6Nakashima K. Sugiura A. Momoi H. Mizuno T. Mol. Microbiol. 1992; 6: 1777-1784Crossref PubMed Scopus (53) Google Scholar) and by partially purified protein after reconstitution (7Nakashima K. Sugiura A. Mizuno T. J. Biochem. (Tokyo). 1993; 114: 615-621Crossref PubMed Scopus (18) Google Scholar). Phosphorylation proceeds most readily in the presence of Mg2+ and at a lower rate in the presence of Ca2+ (6Nakashima K. Sugiura A. Momoi H. Mizuno T. Mol. Microbiol. 1992; 6: 1777-1784Crossref PubMed Scopus (53) Google Scholar). There is moderate homology of parts of the C-terminal domain to other sensor kinases, including His-673. When His-673 was replaced by glutamine, phosphorylation was no longer observed. This finding supports the view that the site of phosphorylation is His-673 (4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). Addition of the soluble response regulator KdpE to phosphorylated KdpD results in rapid transfer of the phosphoryl group to KdpE (4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar, 6Nakashima K. Sugiura A. Momoi H. Mizuno T. Mol. Microbiol. 1992; 6: 1777-1784Crossref PubMed Scopus (53) Google Scholar, 7Nakashima K. Sugiura A. Mizuno T. J. Biochem. (Tokyo). 1993; 114: 615-621Crossref PubMed Scopus (18) Google Scholar). Furthermore, purified KdpE∼P can be dephosphorylated in the presence of everted membrane vesicles containing KdpD (8Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Analysis of a set of KdpD proteins carrying different truncations in the N-terminal part of the protein indicated that in vitro autophosphorylation is dependent on the membrane-spanning domains and that in vivo signal transduction is modulated by the N-terminal region of KdpD (8Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). KdpD presumably functions as a turgor sensor. The turgor control model is based on the pattern of expression of kdpFABC in response to changes in medium K+ concentration and osmolality (9Laimins L.A. Rhoads D.B. Altendorf K. Epstein W. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3216-3219Crossref PubMed Scopus (55) Google Scholar,10Epstein W. Acta Physiol. Scand. 1992; 146: 193-199Google Scholar), reflecting the role of K+ as an important cytoplasmic osmotic solute (11Epstein W. FEMS Microbiol. Rev. 1986; 39: 73-78Crossref Google Scholar). However, further observations lend support to the notion that turgor is apparently not the sole regulatory signal for the expression of kdpFABC and that the availability or intracellular concentration of K+ may also be involved (12Csonka L.N. Hanson A.D. Annu. Rev. Microbiol. 1991; 45: 569-606Crossref PubMed Scopus (641) Google Scholar, 13Asha H. Gowrishankar J. J. Bacteriol. 1993; 175: 4528-4537Crossref PubMed Google Scholar, 14Sugiura A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar). To address the question of the stimulus for KdpD under in vitro conditions and to characterize this protein biochemically, it is necessary to develop an efficient and rapid purification and reconstitution method for KdpD. This report describes the purification of KdpD to >99% homogeneity and an efficient reconstitution method. Purified KdpD in proteoliposomes was tested for its autophosphorylation, phosphotransfer, and dephosphorylation activities. [γ-32P]ATP was purchased from Amersham Buchler. All restriction enzymes and T4 DNA ligase were from New England Biolabs Inc. Taq DNA polymerase was from Life Technologies, Inc. Synthetic oligonucleotide primers were obtained from Eurogentec. Alkaline phosphatase-conjugated goat anti-rabbit IgG was purchased from BIOMOL Research Laboratories Inc. Ni2+-NTA 1The abbreviations used are: NTA, nitrilotriacetic acid; LDAO, lauryldimethylamine oxide; PAGE, polyacrylamide gel electrophoresis; DM, n-dodecyl β-d-maltoside. resin was from QIAGEN Inc. DEAE-Sepharose CL-6B and HiTrap desalting columns were from Pharmacia Biotech Inc. Purified Escherichia coli lipids were purchased from Avanti Polar Lipids. 2-Azido-[α-32P]ATP was a gift from Jan Berden (University of Amsterdam). Centrifugal filter units were purchased from Millipore Corp. Bio-Beads SM-2 were from Bio-Rad. Detergents were obtained from Calbiochem and Anatrace. All other materials were reagent grade and obtained from commercial sources. E. coli JM109 (recA1 endA1 gyrA96 thi hsdR17 supE44 λ − relA1 Δ(lac-proAB)/F′traD36 proA + B + lacIqlacZΔM15) (15Yanisch-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-119Crossref PubMed Scopus (11458) Google Scholar) was used as carrier for the plasmids described. E. coli TKR2000 (ΔkdpFABCDE thi rha lacZ nagA trkA405 trkD1 atp706) (16Kollmann R. Altendorf K. Biochim. Biophys. Acta. 1993; 1143: 62-66Crossref PubMed Scopus (32) Google Scholar) was used for expression of kdpD from the tac promoter. E. coliHAK006 (ΔkdpABCD Δ(lac-pro) ara thi) carries a kdpFABC promoter-lacZ fusion gene and is kdpE + (17Nakashima K. Sugiura A. Kanamaru K. Mizuno T. Mol. Microbiol. 1993; 7: 109-116Crossref PubMed Scopus (55) Google Scholar). This strain was used for determination of in vivo signal transduction. Plasmid pPV5 was used in which kdpD is under the control of thetac promoter (3Walderhaug M.O. Polarek J.W. Voelkner P. Daniel J.M. Hesse J.E. Altendorf K. Epstein W. J. Bacteriol. 1992; 174: 2152-2159Crossref PubMed Google Scholar). To facilitate mutant construction, a unique restriction site (HindIII) after the stop codon of kdpD was created by polymerase chain reaction using plasmid pPV5. Six codons for His were introduced at position 2676 of the kdpD gene by polymerase chain reaction using the mutagenic antisense primer 5′-AA-TCA-GAA-GCT-TTG-TCA-CAT-ATC-ATG-ATG-ATG-ATG-ATG-ATG-CTC-ATG-AAA-TTC-3′ and a sense primer upstream of the ClaI restriction site. The polymerase chain reaction product was purified on agarose gel, digested with ClaI and HindIII, and ligated to similarly treated pPV5, resulting in pPV5-His6. Mutations were verified by sequencing the length of the polymerase chain reaction-generated segment through the ligation junctions in double-stranded plasmid DNA using the dideoxynucleotide termination method (18Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar) and synthetic sequencing primers after alkaline denaturation (19Hattori M. Sakaki Y. Anal. Biochem. 1986; 152: 232-238Crossref PubMed Scopus (1053) Google Scholar). E. coliTKR2000 cells transformed with plasmid pPV5-His6 were grown aerobically at 37 °C in KML complex medium (1% Tryptone, 0.5% yeast extract, and 1% KCl) (20Epstein W. Davies M. J. Bacteriol. 1970; 101: 836-843Crossref PubMed Google Scholar) supplemented with ampicillin (100 μg/ml). Cells were harvested at an absorbance at 600 nm of ∼1.0. Everted membrane vesicles were prepared by passage of cells through a Ribi press and washed twice in an EDTA-containing buffer of low ionic strength (21Siebers A. Altendorf K. Eur. J. Biochem. 1988; 178: 131-140Crossref PubMed Scopus (57) Google Scholar), except that the buffer was changed from Hepes/Tris to Tris/HCl. The washed membrane vesicles were homogenized in 50 mm Tris/HCl, pH 7.5, 10% (v/v) glycerol, and 2 mm dithiothreitol; adjusted to 10 mg/ml protein; and stored until use at −80 °C. To an aliquot (typically 8 ml) of the washed membrane vesicles (5 mg/ml) was added 0.6 m NaCl, and while stirring on ice, KdpD-His6 was extracted with lauryldimethylamine oxide (LDAO) that was added stepwise until a final concentration of 2% (v/v) was reached. After stirring on ice for 30 min, this solubilization mixture was centrifuged for 45 min at 264,000 × g in a Beckman TL100 centrifuge. The supernatant fraction containing solubilized KdpD-His6 was purified by affinity chromatography on a 2-ml column of Ni2+-NTA resin in the following manner. The resin was pre-equilibrated with 25 bed volumes of column buffer (50 mm Tris/HCl, pH 7.5, 10% glycerol, 10 mm β-mercaptoethanol, 0.5 m NaCl, 10 mm imidazole, and 0.04% (w/v) n-dodecyl β-d-maltoside (DM)). Binding of KdpD-His6 was carried out batchwise by incubation of the solubilized proteins and the Ni2+-NTA resin for 30 min at 4 °C. The protein-resin complex was then packed into the column, and unbound protein was removed by washing with column buffer until the absorbance at 280 nm returned to base line. Bound KdpD-His6 was eluted by increasing the imidazole concentration to 100 mm. The flow rate was 1 ml/min, and 2-ml fractions were collected. KdpD-containing fractions were pooled and desalted by passage through a HiTrap desalting column. Final purification of KdpD-His6 was achieved by loading the desalted protein on a 4-ml DEAE-Sepharose CL-6B column pre-equilibrated with 50 mm Tris/HCl, pH 7.5, 10% glycerol, 10 mm β-mercaptoethanol, and 0.04% (w/v) DM. Contaminating proteins were removed by increasing the NaCl concentration stepwise (0, 50, 100, 150, 200, and 300 mm). KdpD-His6 was eluted at 600 mm NaCl. KdpD-His6-containing fractions were pooled and used for reconstitution. Purified KdpD-His6 was reconstituted into E. coliphospholipids essentially as described (22Rigaud J.-L. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (398) Google Scholar). Liposomes were solubilized with the indicated detergents, preferentially DM. The process of liposome solubilization was analyzed by turbidity measurements. In a typical experiment, E. coli phospholipids (5 mg/ml) were solubilized with DM (0.58%, w/v). Then KdpD-His6 in elution buffer was added. The mixture was stirred at room temperature for 10 min. The final ratio of phospholipids to protein was kept at 25 (w/w). Bio-Beads were used to remove the detergent. Bio-Beads were thoroughly rinsed with methanol and buffer (23Holloway P.W. Anal. Biochem. 1973; 53: 304-308Crossref PubMed Scopus (641) Google Scholar) and stored in 50 mm Tris/HCl, pH 7.5, and 10% glycerol at 4 °C until use. To remove the detergents, Bio-Beads at a bead/detergent ratio of 5 (w/w) were added, and the mixture was kept under gentle stirring at 4 °C. After incubation overnight, additional Bio-Beads were added to remove residual detergent. After further incubation for 3 h, the Bio-Beads were pipetted off. The turbid suspension was centrifuged for 1 h at 372,000 × g. The pellet was resuspended in 50 mm Tris/HCl, pH 7.5, 10% glycerol, and 2 mmdithiothreitol. Proteoliposomes were either used instantly or stored in liquid nitrogen. Everted membrane vesicles containing KdpD (2 mg/ml protein), solubilized KdpD (2 mg/ml), or proteoliposomes (0.13 mg/ml) were incubated in phosphorylation buffer containing 50 mm Tris/HCl, pH 7.5, 10% glycerol, 0.5 m NaCl, 10 mmMgCl2, and 2 mm dithiothreitol at room temperature. Phosphorylation was initiated by the addition of 20 μm [γ-32P]ATP (2.38 Ci/mmol) or 2 μm [γ-32P]ATP (23.8 Ci/mmol) as indicated. At different times, aliquots were removed and mixed with an equal volume of twice concentrated SDS sample buffer (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). After incubation for 5 min, an equimolar amount of purified KdpE (16.5 μg/ml) (8Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) was added to the KdpD-containing fractions, and the incubation was continued. Additional aliquots were removed at different times and mixed with SDS sample buffer as described above. To test dephosphorylation, purified KdpE was phosphorylated in the following manner. Wild-type KdpD (0.67 mg/ml) was incubated in phosphorylation buffer containing 5 mm CaCl2instead of MgCl2, and phosphorylation was initiated with 2 μm [γ-32P]ATP (23.8 Ci/mmol). After 10 min, purified KdpE (16.5 μg/ml) was added, and the incubation was continued for 5 min. KdpD-containing membrane vesicles were removed by two filtration steps. First, a membrane with a pore size of 0.22 μm was used, followed by a second filtration through a low protein-binding membrane that cuts off proteins with a M r of >30,000. ATP was removed by the addition of 20 mmMgCl2, 3.2 mm glucose, and 5.4 units of hexokinase. Dephosphorylation was initiated by the addition of KdpD-His6 in everted membrane vesicles (0.44 mg/ml) or proteoliposomes (0.09 mg/ml). At the times indicated, aliquots were removed, and the reaction was stopped by the addition of SDS sample buffer. All samples were immediately subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar). Gels were dried, and phosphorylation of the proteins was detected by autoradiography with Eastman Kodak BIOMAX MR film for 14 h at room temperature. Binding of 2-azido-[α-32P]ATP was studied with everted membrane vesicles containing KdpD-His6 (1 mg/ml) or solubilized KdpD-His6 (1 mg/ml) in different detergents. The samples were preincubated in 50 mm Tris/HCl, pH 7.5, 10% glycerol, and 2 mm dithiothreitol. The reaction was initiated by the addition of 52 μm 2-azido-[α-32P]ATP (0.34 Ci/mmol) and incubated for 3 min. Cross-linking was achieved by irradiation of the sample with UV light at 302 nm for 2.5 min. The same experiment was repeated except that the samples were preincubated with an excess of ATP (10 mm) for 3 min before 2-azido-[α-32P]ATP was added. All samples were immediately subjected to SDS-PAGE. Gels were dried, and phosphorylation of the proteins was detected by autoradiography. For Western blot analysis, everted membrane vesicles or proteoliposomes were solubilized in an equal volume of twice concentrated SDS sample buffer. Equal amounts of protein were subjected to SDS-PAGE. Proteins were electroblotted, and immunoblots were probed with polyclonal antibodies against KdpD as described (4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). Immunodetection was performed using an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (4Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). Proteoliposomes (0.2 mg/ml) in the corresponding buffer were incubated with kallikrein (100 μg/ml) for 1 h at 37 °C. At the indicated times, the reaction was stopped by the protease inhibitor leupeptin (1 μm), and the protein was subjected to SDS-PAGE and silver-stained. In vivosignal transduction was probed with E. coli HAK006 transformed with the plasmids described. Cells were grown in TY medium (1% Tryptone and 0.5% yeast extract) (14Sugiura A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar) supplemented with 0.6m NaCl and KCl as indicated. The basal concentration of K+ in the medium was determined to be 6 mm by flame photometry. Cells were grown to the mid-logarithmic growth phase and harvested by centrifugation. The cell pellet was resuspended in 200 mm sodium phosphate buffer, pH 7.2. An aliquot of this suspension was used to determine the absorbance at 600 nm, and another aliquot was used for determination of β-galactosidase activity after the cells were permeabilized with toluene. β-Galactosidase activity was determined from at least three different experiments and is given in Miller units calculated as described (25Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972Google Scholar). The turbidity of the phospholipid vesicle suspension as a function of detergent concentration was measured at an absorbance of 600 nm with a Shimadzu spectrophotometer. Liposomes were suspended at a concentration of 5 mg/ml. The detergent concentration was raised in increments, and absorbance was continuously recorded until a steady-state level was reached. Protein was assayed by a modified version (26Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7127) Google Scholar) of the method of Lowry et al.(45Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Proteins were separated by SDS-PAGE (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207018) Google Scholar) using 7.5, 9, 11, and 12.5% acrylamide gels. Silver staining was performed as described (27Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3739) Google Scholar). Oligonucleotide-directed, site-specific mutagenesis was used to insert six consecutive triplets encoding histidine two triplets before the stop codon of kdpD (Fig.1). This insertion was verified by double-stranded DNA sequencing. The gene encoding the tagged sensor kinase was cloned in pPV5 and overexpressed under the control of the tac promoter in E. coli TKR2000. KdpD-His6 was detectable in everted membrane vesicles in amounts that are comparable to wild-type KdpD as judged from immunoblot analysis (data not shown). KdpD-His6 in everted membrane vesicles was able to undergo autophosphorylation in the presence of ATP. After the addition of the purified response regulator KdpE, a rapid transfer of the phosphoryl group from phosphorylated KdpD-His6 to KdpE occurred (data not shown). In addition, KdpD-His6 caused dephosphorylation of KdpE∼P in a time-dependent manner. Simultaneously with the decrease in the amount of KdpE∼P, an increase in the amount of phosphorylated KdpD-His6 was observed (data not shown). All activities tested with KdpD-His6 were indistinguishable from those of wild-type KdpD. Furthermore, the sensing and signal transduction capacity of KdpD-His6 in vivo was tested. For this purpose,E. coli HAK006 was used (14Sugiura A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar), which lacks the functionalkdpFABC operon as well as kdpD on the chromosome, but contains the intact kdpE gene under the control of its own promoter. This strain carries a kdpFABCpromoter-lacZ fusion gene on the chromosome. Upon transformation of this strain with a plasmid carrying the intactkdpD gene, the inducible expression of thekdpFABC operon can be probed by measuring β-galactosidase activity. It has been found previously that the extent ofkdpFABC expression is dependent on the K+concentration and the osmolality of the medium (9Laimins L.A. Rhoads D.B. Altendorf K. Epstein W. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3216-3219Crossref PubMed Scopus (55) Google Scholar). E. coliHAK006 cells were transformed with plasmid pPV5 or pPV5-His6 and grown in a medium containing 0.6m NaCl and only basal levels of K+. The steady-state β-galactosidase activity of these cells was high (110 units), indicating that KdpD as well as KdpD-His6 were able to respond to high osmolality. Moreover, β-galactosidase activity was reduced to basal levels (0.8 units) when the cells were cultivated in a medium of the same osmolality containing 40 mmK+, which was sufficient to repress expression ofkdpFABC. Thus, a significant difference between the activities of KdpD-His6 and wild-type KdpD was observed neither under in vivo nor under in vitroconditions. Efficient solubilization of the membrane-bound histidine kinase is one of the prerequisites for purification. To select a detergent meeting this criterion, everted membrane vesicles were incubated in the presence of different detergents at final concentrations exceeding their critical micelle concentrations. After a 30-min incubation on ice, the reaction mixture was centrifuged, and aliquots of the supernatant and the resuspended membrane pellet were analyzed by immunoblotting (data not shown). The zwitterionic detergents LDAO, Zwittergent 3-12, and Zwittergent 3-14 were found to be the most efficient detergents in solubilizing KdpD-His6. Furthermore, solubilization of KdpD-His6 could be increased to nearly 100% when the solubilization was carried out in the presence of NaCl. Nonionic detergents had limited solubilization efficiency. The efficiency increased with the length of the carbon chain: octyl glucoside < decyl maltoside < DM. Maximal solubilization with DM was ∼50%. When KdpD-His6 solubilized in LDAO, DM, or Zwittergent 3-12 was tested for autophosphorylation activity, no phosphorylation of the protein was detectable (data not shown). In addition to standard conditions, phosphorylation was also tested in the presence of different NaCl concentrations (0, 50, and 150 mm). The ATP concentration was also varied. However, no significant phosphorylation of KdpD-His6 was observed under any of the conditions tested (data not shown). Furthermore, the ability of solubilized KdpD-His6 to bind 2-azido-[α-32P]ATP was analyzed. As shown in Fig.2, KdpD-His6 solubilized in DM was able to bind 2-azido-ATP. As a comparison, binding of 2-azido-ATP to KdpD-His6 in everted membrane vesicles is shown. More important, 2-azido-ATP binding could not be observed when KdpD-His6 was solubilized in LDAO or Zwittergent 3-12 (data not shown). Since solubilization of KdpD-His6 from membranes was nearly 100% using the detergent LDAO in the presence of NaCl, this detergent was used for the purpose of purification. Taking into account that only DM-solubilized KdpD-His6 was able to bind 2-azido-ATP, the detergent was changed from LDAO to DM in the first column chromatography step. The protocol used for the purification of KdpD-His6 comprised two steps: purification by nickel chelate affinity chromatography and ion exchange chromatography. Experiments were generally carried out starting from 40 mg of protein of everted membrane vesicles. Progress of the purification was judged by SDS-PAGE analysis (Fig.3). In a typical experiment, everted membrane vesicles were solubilized in the presence of 2% LDAO and 0.6 m NaCl as described under “Experimental Procedures.” As judged by the protein content, solubilization with LDAO was not very selective. Thus, 90% of the membrane proteins were solubilized by this detergent. The highest degree of purification of KdpD-His6 was achieved by the nickel chelate chromatography. Binding of KdpD-His6 to the Ni2+-NTA resin was most effective when the solubilized proteins were incubated batchwise with pre-equilibrated resin for 30 min. Furthermore, selective binding of KdpD-His6 was greatly enhanced by the addition of low amounts of imidazole (10 mm) and relatively high amounts of NaCl (0.5m). When the absorbance at 280 nm of the elute returned to base line, bound KdpD-His6 was eluted with 100 mm imidazole. As demonstrated by silver staining (Fig. 3), the fraction eluted with 100 mm imidazole shows a major protein band at ∼98 kDa that corresponds to KdpD-His6 and some other minor bands. To remove the residual contaminating proteins, ion exchange chromatography was used as a last purification step. Before loading the protein on a DEAE-Sepharose CL-6B column, the ionic strength of the pooled Ni2+-NTA column fractions was lowered by passage through a HiTrap desalting column. KdpD-His6 was bound to the ion exchanger. By applying a stepwise gradient of NaCl, contaminating proteins were eluted, whereas KdpD-His6 still remained bound to the resin. Finally, KdpD-His6 could be eluted at a NaCl concentration of 0.6 m. The eluted protein was subjected to SDS-PAGE, and after silver staining, only one band was detectable (Fig. 3). This protein band was identified as KdpD by Western blot analysis (Fig. 3). KdpD-His6 appeared as a single band; no dimer formation or even higher order aggregates were observed. Overall, starting from 10 g of cells (wet weight), 1.5 mg of purified KdpD-His6 was obtained. Fractions containing KdpD-His6 eluted with 0.6 m NaCl from the DEAE-Sepharose column were pooled, and the protein was reconstituted into E. coli phospholipids. Reconstitution was carried out using the detergent-mediated method as described (22Rigaud J.-L. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (398) Google Scholar). Liposomes were solubilized with increasing concentrations of DM, and the process of liposome solubilization was followed by turbidity measurements. As described for other membrane proteins, the stage of solubilization of liposomes prior to the addition of solubilized protein results in proteoliposomes of different activity (22Rigaud J.-L. Pitard B. Levy D. Biochim. Biophys. Acta. 1995; 1231: 223-246Crossref PubMed Scopus (398) Google Scholar). For DM, two characteristic solubilization stages of the liposomes were tested: partial solubilization and total solubilization. Purified KdpD-His6in elution buffer was added to the solubilized liposomes, and the detergent was removed by the addition of Bio-Beads. Upon reconstitution of KdpD-His6 into proteoliposomes, autophosphorylation activity was recovered (Fig. 4 A). The highest phosphorylation activity was achieved with liposomes that were totally solubilized with DM, as shown in Fig. 4 A. Additional detergents were tested for their ability to mediate reconstitution of KdpD-His6. Thus, LDAO, Triton X-100, sodium cholate, and octyl glucoside were used, and different stages of liposome solubilization were tested. Although all prepared KdpD-His6-containing proteoliposomes exhibited autophosphorylation activity (data not shown), the highest activity was always observed with liposomes that were solubilized with DM. The efficiency of KdpD-His6 reconstitution was determined to be 35%. In addition to testing the autophosphorylation activity of purified KdpD-His6 in proteoliposomes, transfer of the phosphoryl group to purified KdpE was examined. Therefore, proteoliposomes were incubated with [γ-32P]ATP, and after 5 min, an equimolar amount of purified response regulator KdpE was added. Subsequent transfer of the phosphoryl group to KdpE occurred (Fig. 4 A). To determine whether purified KdpD-His6 would regenerate KdpE from its phosphorylated form, KdpE∼P was incubated with KdpD-His6 in proteoliposomes. A time-dependent dephosphorylation of KdpE∼P was observed (Fig. 4 B). Dephosphorylation of KdpE∼P was accompanied by an increase in the amount of phosphorylated KdpD-His6 (Fig. 4 B). The accessibility of ATP to KdpD-His6 in proteoliposomes suggested already an inside-out orientation of this protein. To obtain additional evidence for this, the susceptibility to the protease kallikrein was tested. In both the N- and C-terminal regions of KdpD, but not in the central hydrophobic region, 10 potential cleavage sites for kallikrein can be found. Earlier studies revealed that treatment of spheroplasts with kallikrein did not affect the kinase at all, but after permeabilization of the spheroplasts, KdpD was degraded completely, indicating that the protease cleavage sites are accessible only from inside the cells (5Zimmann P. Puppe W. Altendorf K. J. Biol. Chem. 1995; 270: 28282-28288Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). When proteoliposomes were incubated with kallikrein, KdpD-His6 was completely degraded (Fig.5). This result provides strong evidence for the unidirectional inside-out orientation of KdpD-His6 in proteoliposomes. Most histidine kinase molecules in bacteria, including KdpD, are integral membrane proteins (28Parkinson J.S. Kofoid E.C. Annu. Rev. Genet. 1992; 26: 71-112Crossref PubMed Scopus (1242) Google Scholar, 29Swanson R.V. Bourret R.B. Simon M.I. Mol. Microbiol. 1993; 8: 435-441Crossref PubMed Scopus (103) Google Scholar). To study their activity in vitro, it is therefore necessary to establish an efficient solubilization and purification protocol. In the case of KdpD, this was achieved by applying the histidine peptide fusion technique (30Ljungquist C. Breitholtz A. Brink-Nilsson H. Moks T. Uhlé M. Nilsson B. Eur. J. Biochem. 1989; 186: 563-569Crossref PubMed Scopus (89) Google Scholar). Although the His tag is located in the C-terminal domain, which is most certainly responsible for the interaction with ATP and the cytoplasmic response regulator KdpE, no significant changes with respect to phosphorylation, phosphotransfer, or dephosphorylation in comparison to the wild type could be observed. In addition, sensing and signal transduction in vivo were not influenced by the insertion of six His residues in KdpD as shown by the pattern of expression of thekdpFABC promoter-lacZ fusion gene in E. coli HAK006. To our knowledge, there is, so far, no other example of a fusion at the C-terminal end of a sensor kinase. In contrast, different sensor kinases with modifications at the N terminus have been described. Thus, the soluble parts of the sensor kinases NarX and NarQ were fused at the N-terminal end with six His residues, and the purified proteins were active (31Schröder I. Wolin C.D. Cavicchioli R. Gunsalus R.P. J. Bacteriol. 1994; 176: 4985-4992Crossref PubMed Google Scholar). In NRII, the N-terminal domain was replaced by the maltose-binding protein. This protein still catalyzed autophosphorylation and transfer of the phosphoryl group to the response regulator (NRI), but it was unable to stimulate the dephosphorylation of NRI∼P (32Kamberov E.S. Atkinson M.R. Chandran P. Ninfa A.J. J. Biol. Chem. 1994; 269: 28294-28299Abstract Full Text PDF PubMed Google Scholar). Solubilization of KdpD-His6 in different detergents resulted in an inactive protein. This result is in agreement with earlier attempts to detect autophosphorylation activity of the solubilized protein (7Nakashima K. Sugiura A. Mizuno T. J. Biochem. (Tokyo). 1993; 114: 615-621Crossref PubMed Scopus (18) Google Scholar). Obviously, only the membrane-bound protein exhibits autophosphorylation activity. This assumption is further supported by the analysis of a number of truncated KdpD proteins (8Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Different truncations of the N-terminal part resulted in KdpD proteins that retain autophosphorylation and phosphotransfer activities in vitro. In contrast, phosphorylation could not be detected in those forms of KdpD in which the membrane-spanning segments were removed. It is important to note that these inactive truncated forms of KdpD could be labeled with 2-azido-[α-32P]ATP, indicating that binding of ATP to KdpD was not affected by removal of the hydrophobic domain. Our studies demonstrate that DM-solubilized KdpD-His6 also retains its ability to bind 2-azido-ATP. This observation revealed that binding of ATP is not dependent on the anchoring of the protein in the membrane. In contrast, 2-azido-ATP binding was not detectable when KdpD-His6 was solubilized in zwitterionic detergents. Obviously, DM stabilizes the structure of KdpD, an effect that was also observed for other membrane proteins (e.g. Ref. 33Wu J. Kaback H.R. Biochemistry. 1994; 33: 12166-12171Crossref PubMed Scopus (81) Google Scholar). Reconstitution of KdpD-His6 into proteoliposomes restores full activity of the sensor kinase. Therefore, KdpD-His6 in proteoliposomes was phosphorylated when incubated in the presence of ATP, and the phosphoryl group could be transferred to the soluble response regulator KdpE. That these activities of KdpD are dependent on a membranous environment is atypical for membrane-bound sensor kinases. Soluble truncated forms of the sensor kinases FixL (34Gilles-Gonzalez M.A. Ditta G.S. Helinski D.R. Nature. 1991; 350: 170-172Crossref PubMed Scopus (417) Google Scholar), EnvZ (35Forst S.A. Delgado J. Inouye M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6052-6056Crossref PubMed Scopus (193) Google Scholar, 36Roberts D.L. Bennett D.W. Forst S.A. J. Biol. Chem. 1994; 269: 8728-8733Abstract Full Text PDF PubMed Google Scholar), and NarX and NarQ (31Schröder I. Wolin C.D. Cavicchioli R. Gunsalus R.P. J. Bacteriol. 1994; 176: 4985-4992Crossref PubMed Google Scholar) are capable of undergoing autophosphorylation. The finding that anchoring of KdpD in the membrane is a prerequisite for an active conformation is in accordance with the current model for the regulation of kdpFABC expression (1Altendorf K. Epstein W. Lee A.G. Biomembranes.5. Jai Press, Inc., London, England1996: 403-420Google Scholar). When turgor (and hence, membrane stretch) is high, KdpD is inactive. When turgor is reduced, KdpD alters its conformation, and the kinase becomes active, resulting in the formation of KdpE∼P and thus expression ofkdpFABC. However, it is also conceivable that the key feature of regulation is the dephosphorylation of KdpE∼P. Purified and reconstituted KdpD-His6 is able to facilitate dephosphorylation of purified KdpE∼P. Thus, dephosphorylation of KdpE∼P is dependent only on the histidine kinase KdpD. Dephosphorylation of the response regulator has been well studied in other sensor kinase/response regulator systems and characterized by diverse mechanisms (37Stock J.B. Surette M.G. Levit M. Park P. Hoch J.A. Silhavy T.J. Two-Component Signal Transduction. American Society for Microbiology, Washington, D. C.1995: 25-51Google Scholar). The sensor kinase EnvZ by itself is able to enhance the rate of dephosphorylation of phospho-OmpR (38Igo M.M. Ninfa A.J. Stock J.B. Silhavy T.J. Genes Dev. 1989; 3: 1725-1734Crossref PubMed Scopus (218) Google Scholar, 39Aiba H. Nakasai F. Mizushima S. Mizuno T. J. Biol. Chem. 1989; 264: 14090-14094Abstract Full Text PDF PubMed Google Scholar). In the case of NRII, an auxiliary protein (PII) is involved (32Kamberov E.S. Atkinson M.R. Chandran P. Ninfa A.J. J. Biol. Chem. 1994; 269: 28294-28299Abstract Full Text PDF PubMed Google Scholar, 40Keener J. Kustu S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4976-4980Crossref PubMed Scopus (233) Google Scholar, 41Ninfa A.J. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5909-5913Crossref PubMed Scopus (386) Google Scholar). In the chemotaxis system, dephosphorylation of CheY is mediated by a separate protein, CheZ (42Blat Y. Eisenbach M. Biochemistry. 1994; 33: 902-906Crossref PubMed Scopus (63) Google Scholar), and in the process of sporulation of Bacillus subtilis, different phosphatases have been identified (43Perego M. Hanstein C. Welsh K.M. Djavakhishvili T. Glaser P. Hoch J.A. Cell. 1994; 79: 1047-1055Abstract Full Text PDF PubMed Scopus (250) Google Scholar). Dephosphorylation of KdpE∼P is accompanied by an increase in the amount of phosphorylated KdpD. Whether this is due to a reverse phosphotransfer of the phosphoryl group from KdpE to KdpD or to residual amounts of ATP is still under investigation. For the EnvZ/OmpR system, a reverse phosphotransfer of the phosphoryl group from OmpR to the autophosphorylation site of a kinase−/phosphatase+ mutant of EnvZ was recently reported (44Dutta R. Inouye M. J. Biol. Chem. 1996; 271: 1424-1429Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In summary, we have shown that the purified and reconstituted KdpD-His6 protein retains enzymatic activity. KdpD has an autokinase activity, and the phosphoryl group can be transferred to the response regulator KdpE. Finally, KdpD facilitates the dephosphorylation of purified KdpE∼P. This study provides the basis for further analyses of the structure and function of KdpD. In this context, the question of how KdpD senses changes in turgor is especially interesting. We thank Dr. T. Mizuno for providing E. coli strain HAK006 and M. Lucassen for providing purified response regulator KdpE." @default.
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• W2074001058 title "Purification, Reconstitution, and Characterization of KdpD, the Turgor Sensor of Escherichia coli" @default.
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