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J Biol Chem, Vol. 275, Issue 9, 6241-6245, March 3, 2000

Identification and Characterization of a Novel cAMP Receptor Protein in the Cyanobacterium Synechocystis sp. PCC 6803^*

Hidehisa Yoshimura, Toru Hisabori^§, Shuichi Yanagisawa, and Masayuki Ohmori^¶

From the  Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro, Tokyo 153-8902 and the ^§ Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuda 4259, Midori-ku, Yokohama 226-8503, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Three open reading frames of Synechocystis sp. PCC 6803 encoding a domain homologous with the cAMP binding domain of bacterial cAMP receptor protein were analyzed. These three open reading frames, sll1371, sll1924, and slr0593, which were named sycrp1, sycrp2, and sypk, respectively, were expressed in Escherichia coli as His-tagged or glutathione S-transferase fusion proteins and purified, and their biochemical properties were investigated. The results obtained for equilibrium dialysis measurements using these recombinant proteins suggest that SYCRP1 and SYPK show a binding affinity for cAMP while SYCRP2 does not. The dissociation constant of His-tagged SYCRP1 for cAMP is approximately 3 µM. A cross-linking experiment using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide revealed that His-tagged SYCRP1 forms a homodimer, and the presence or absence of cAMP does not affect the formation of the homodimer. The amino acid sequence reveals that SYCRP1 has a domain similar to the DNA binding domain of bacterial cAMP receptor protein in the COOH-terminal region. Consistent with this, His-tagged SYCRP1 forms a complex with DNA that contains the consensus sequence for E. coli cAMP receptor protein in the presence of cAMP. These results strongly suggest that SYCRP1 is a novel cAMP receptor protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Cyclic AMP (cAMP) is a global signaling molecule that exists in both prokaryotes and eukaryotes. It mediates a signal from outside the cell to a target protein that regulates gene expression or the enzyme activity of various enzymes. cAMP plays a central role in the regulation of the response to different nutritional states in enteric bacteria (1). Glucose is known to lower intracellular cAMP levels under certain conditions in Escherichia coli (2, 3). cAMP functions to regulate transcription in conjunction with the cAMP receptor protein (CRP)¹ in Gram-negative bacteria. E. coli CRP has been well characterized in vitro by a variety of physical and biochemical techniques, including x-ray analysis of CRP crystals (4, 5). The CRP forms a homodimer; each subunit of this dimer is composed of two domains. The larger NH₂-terminal domain contains the cAMP binding site and shows homology to a class of cAMP-dependent protein kinases (6-8). The dimer form of CRP is able to bind to specific DNA sequences when it is liganded with cAMP. The COOH-terminal domain can bind to DNA at helix-turn-helix motif. The cAMP-CRP complex functions as a transcriptional regulator, activating transcription at several promoters and repressing transcription from others (1, 9-12).

In the green algae Chlamydomonas, cAMP is reported as a key second messenger in the mating reaction, but the adenylate cyclase and the mediator of cAMP have not yet been identified. In this case, the cAMP signal cascade is poorly understood (13). In Euglena, Carrie and Edmunds (14) have reported that cAMP mediates the phasing of the cell division cycle through the circadian clock. Recently, it was reported that the catalytic subunit of PKA had been identified in Euglena gracilis (15), although the adenylate cyclase and regulatory subunit of PKA have not been reported. In higher plants, many attempts have been made to clone the adenylate cyclase and PKA genes. Nevertheless, these genes have not been identified in higher plants.

Cyanobacteria, Gram-negative bacteria, are able to perform higher plant-type oxygen evolving photosynthesis. The intracellular cAMP levels of cyanobacteria change in response to changes in environmental conditions such as light-dark, low pH-high pH, oxic-anoxic (16, 17), and nitrogen replete-deplete (18). Exogenous cAMP stimulates the gliding movement of Spirulina platensis (19) and also enhances the cell motility of Synechocystis sp. PCC 6803 (20). These results suggest that cAMP functions as a signaling molecule in cyanobacteria as well. However, no intracellular molecule that mediates the cAMP signal has yet been identified in cyanobacteria.

In this report, we investigated the biochemical properties, especially the cAMP binding affinity, of putative cyanobacterial CRPs. Among them, a novel cAMP receptor protein was identified as a cyanobacterial cAMP receptor protein and named SYCRP1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Bacterial Strains and Growth Media-- The E. coli strains used as hosts were JM109 (recA1, endA1, gyrA96, thi, hsdR17(rK, mK⁺)), supE44, relA1, (lac-proAB)/F' (traD36, proAB, lacI^q, (lacZ)M15)) for cloning and BL21(DE3)pLysS (F, ompT, hsdS (rB, mB), dcm, gal, (DE3), pLysS) for the expression of recombinant proteins. Bacteria were grown in Luria-Bertani medium (21). When required, kanamycin or chloramphenicol was added at 25 or 30 µg ml¹, respectively.

Construction of Expression Plasmids for Recombinant Proteins-- To obtain DNA fragments corresponding to sll1371, sll1924, and slr0593 ORFs (sycrp1, sycrp2 and sypk, respectively), polymerase chain reactions were performed with several sets of synthetic primers and genomic DNA from Synechocystis sp. PCC 6803. The primers for sycrp1 were CGA1 (5'-CGGGATCCATGGGCACTAGTCCCC-3') and CGA2 (5'-CCCACCTAGTGTGACAT-3'). Primers for sycrp2 were HYP1(5'-CGGGATCCATGGCACCACAAAAGC-3') and HYP2 (5'-GGCAACTGCTGAGGTTT-3'). Primers for sypk were PKA1 (5'-CGGGATCCATGGAACTGGGAAAGATT-3') and PKA2 (5'-CAGGCTCTTAGACGTGG-3'). The sequences of the CGA1, HYP1, and PKA1 primers were designed to allow the introduction of a BamHI restriction site immediately upstream of the initiator ATG codon. Each polymerase chain reaction product was cloned into pCRII vector (Invitrogen). After verification of the nucleotide sequence, sycrp1 and sycrp2 were digested from each plasmid with BamHI and EcoRI and cloned between the BamHI and EcoRI sites of the pET-28a expression vector (Novagen). The resulting plasmids were named pCGA and pHYP. The sypk was partially digested with AluI, and a DNA fragment corresponding to a putative cAMP binding site (from 291 to 872 relative to the start site of the open reading frame) was cloned into the SmaI site of pGEX-2T (Amersham Pharmacia Biotech). The direction of the open reading frame was confirmed by restriction mapping. The resulting plasmid was named pPKA.

Expression and Purification of Recombinant Proteins-- The transformants, BL21(DE3)pLysS cells harboring each of the pCGA, pHYP, and pPKA plasmids, were grown at 37 °C in 1.5 liter of Luria-Bertani medium supplemented with kanamycin or ampicillin (25 or 100 µg ml¹, respectively) and chloramphenicol (30 µg ml¹). The recombinant genes were expressed in exponentially growing cells by adding 1 mM isopropyl--D-thiogalactopyranoside (IPTG). After 3 h of incubation, the cells were harvested by centrifugation, washed with 50 mM Tris-HCl (pH 8.0) buffer containing 100 mM NaCl, and resuspended in 50 ml of Buffer A consisting of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 10% (w/v) glycerol. The cell suspension was frozen and thawed, and then sonicated at 4 °C for 9 min (3 min three times) using a sonicator (model 200M, Kubota Co., Tokyo, Japan). The cell extract was centrifuged at 16,000 × g for 10 min, and the supernatant was further centrifuged at 150,000 × g for 45 min.

Recombinant proteins in the 150,000 × g supernatants were purified with several affinity columns connected to a fast protein liquid chromatography system (FPLC system, Amersham Pharmacia Biotech). The 150,000 × g supernatant containing His-tagged SYCRP1 (His-SYCRP1) was loaded onto a HiTrap chelating column (Amersham Pharmacia Biotech; 1.6 × 2.5 cm) connected to an FPLC system, and the proteins were eluted using a step gradient of 30, 60, 100, and 200 mM imidazole in Buffer A. The 200 mM imidazole fraction was loaded onto a HiTrap Q column (Amersham Pharmacia Biotech; 1.6 × 2.5 cm) connected to an FPLC system, and eluted using a step gradient of 100 and 200 mM NaCl in Buffer B consisting of 50 mM Tris-HCl (pH 8.0) and 10% (w/v) glycerol. The 200 mM NaCl fraction contained purified His-SYCRP1. The predicted molecular mass of this protein was determined to be 29.5 kDa using DNASTAR software (DNASTAR Inc).

The 150,000 × g supernatant containing His-tagged SYCRP2 (His-SYCRP2) was loaded onto a HiTrap SP column (Amersham Pharmacia Biotech; 0.7 × 2.5 cm) connected to an FPLC system, and was eluted using a step gradient of 100, 200, and 400 mM NaCl in Buffer B. The 400 mM NaCl fraction was loaded onto a HiTrap chelating column connected to an FPLC system, and eluted using a step gradient of 100, 200, and 400 mM imidazole in Buffer A. The 400 mM imidazole fraction contained purified His-SYCRP2. The predicted molecular mass of His-SYCRP2 is 30.4 kDa.

The 150,000 × g supernatant containing a fusion protein (GST-pSYPK) between GST and a portion of SYPK was loaded onto a glutathione-Sepharose 4B column (10 ml, Amersham Pharmacia Biotech), and eluted with 5 mM glutathione in Buffer A. The eluate was loaded onto a HiTrap Q column connected to an FPLC system, and eluted using a step gradient of 100 and 200 mM NaCl in Buffer B. The 200 mM NaCl fraction contained purified GST-pSYPK. The predicted molecular mass of GST-pSYPK is 45.5 kDa.

Equilibrium Dialysis-- Equilibrium dialysis was performed essentially as described by Hisabori et al. (22). Lucite cells were fulfilled in a volume of 100 µl per half cell with dialysis membranes (pore size 15 Å; Biomed Instruments Inc. Co., Fullerton, CA). A glass bead (inner diameter = 0.8 mm) was placed in each half cell to aid stirring. One of the recombinant proteins in 100 µl of 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 10% (w/v) glycerol was placed in one side (sample side) of the lucite cell, and the same volume of cAMP solution containing 50 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 10% (w/v) glycerol was placed in the other side (reference side). The concentrations of the recombinant proteins and cAMP are indicated in the legends to Figs. 3 and 4. The filled lucite cells were incubated in a shaker (BR-15LF, Taitec Co., Saitama, Japan) at 20, 25, or 30 °C. An incubation period of 6 h was needed to establish equilibria, and 80-µl samples were taken from each half cell for HPLC analysis.

Assay for Bound cAMP by Reversed-phase HPLC-- An HPLC system (Shimadzu Co., Kyoto, Japan) was used to measure the amounts of cAMP bound to recombinant proteins. Aliquots from the sample side were denatured for 5 min with cold trichloroacetic acid, final concentration 4.5% (w/v), or by heating at 95 °C for 2 min, and then centrifuged at 18,000 × g for 5 min at 4 °C to remove proteins. The 10-µl aliquot of the supernatant from the sample side or 10 µl from the reference side was applied to a TSK ODS-80Ts column (4.6 mm × 15 cm, Tosoh Co., Tokyo, Japan) equilibrated with 30 mM sodium phosphate buffer (pH 6.5) containing 5% (v/v) acetonitrile; the effluent was monitored at 259 nm. The flow rate was 1.0 ml/min.

Cross-linking of His-SYCRP1 with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-- Purified His-SYCRP1 (4 µg) was incubated in 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, and 10% (w/v) glycerol, with or without 10 µM cAMP in a final volume of 20 µl for 10 min at room temperature prior to the addition of EDC to a final concentration of 25 mM. After 2 h of incubation, the cross-linking reaction was stopped by the addition of 4× SDS sample-loading buffer. The samples were loaded onto a 10% SDS-PAGE gel for electrophoresis. The gel was stained with Coomassie Brilliant Blue R-250.

Gel Mobility Shift Assay-- An oligonucleotide (5'-CGGGATCCGCGAAAAGTGTGACATATGTCACACTTTTCGC-3') was synthesized and used in the assay. The sequence of the oligonucleotide includes a consensus DNA sequence for E. coli CRP binding and a BamHI sequence attached to the 5' end of the consensus sequence. Since the consensus sequence is composed of two palindromic copies of a 16-bp sequence, preparation of double strand DNA was achieved by incubating the oligonucleotide at 95 °C for 1 min, 60 °C for 10 min, and 25 °C for 1 h in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM dithiothreitol. The DNA fragment was labeled with Klenow fragment and [-³²P]dCTP (ICN Biomedicals Inc., Costa Mesa, CA), and used as a probe. The ³²P-labeled probe (10,000 cpm, approximately 1 ng) was incubated with His-SYCRP1 in a total volume of 20 µl of the binding buffer (50 mM Tris-HCl (pH 7.5), 60 mM NaCl, 1 mM EDTA, 8% (w/v) glycerol, 50 ng of poly(dI-dC)) with or without a final concentration of 20 µM cAMP for 30 min at room temperature. Samples were loaded onto 5% polyacrylamide gels (acrylamide:N,N'-methylenebisacrylamide, 50:1). The electrophoresis buffer was 0.25× TBE with or without 20 µM cAMP. Electrophoresis was conducted at constant current (15 mA). When the binding reactions were carried out in buffer containing cAMP, electrophoresis buffer supplemented with 20 µM cAMP was also used. After electrophoresis, the gels were dried and autoradiographed.

Other Analytical Procedures-- Protein concentration was determined by the method of Bradford (23). Bovine serum albumin was used as the standard. SDS-PAGE was carried out in polyacrylamide gels containing 0.1% SDS by the method of Laemmli (24).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Selection of ORFs That Predictably Encode cAMP Receptor Proteins-- The cAMP binding motif in the prokaryotic cAMP receptor protein and the eukaryotic regulatory subunit of cAMP-dependent protein kinase is substantially conserved (6-8). We performed a search for a putative cAMP receptor protein in the Synechocystis PCC 6803 genome sequencing data (25) and Cyano Base, with reference to the entire amino acid sequence of E. coli CRP, and 18 candidate ORFs were first selected. Based on the presence of functional amino acids that bind cAMP molecule (4, 26), 18 ORFs were finally narrowed down to 3 ORFs (Fig. 1): sll1371, sll1924, and slr0593, which we named sycrp1, sycrp2 and sypk, respectively.

View larger version (57K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence alignment of E. coli CRP, Synechocystis sp. PCC 6803 SYCRP1, SYCRP2, and SYPK. The asterisks mark amino acid residues that lie close to the cAMP molecule, open stars indicate residues that function in binding the cAMP molecule to the E. coli CRP structure, and open triangles indicate residues that are identical in all four proteins. Identical and similar residues in at least three of the sequences are shown in gray boxes.

Comparison of Deduced Amino Acid Sequences of Three ORFs with E. coli CRP Sequence-- The predicted amino acid sequences of the three ORFs are overall less similar to E. coli CRP sequence (Fig. 1). The residue identity is only 14-23% between three proteins and E. coli CRP, although some highly conserved amino acid residues were identified among these amino acid sequences. The amino-terminal region, from residues 1 to 88 of SYCRP1, shows low sequence homology to the corresponding region of E. coli CRP. However, the next segment, corresponding to residues 89-103 of SYCRP1, shows high homology to E. coli CRP and contains several amino acids that participate directly in the binding of cAMP (indicated by the open stars in Fig. 1). The identical residues in this segment are Gly-89, Glu-90, Glu-95, Glu-96, Arg-99, Ser-100, and Val-103, which correspond to Gly-72, Glu-73, Glu-78, Glu-79, Arg-83, Ser-84, and Val-87 in E. coli CRP, respectively. The next highly homologous region, residues 138-141 of SYCRP1, is highly conserved. Arg-124 stabilizes Glu-73 in E. coli CRP through an ionic interaction (4, 26). Thr-128 and Ser-129 participate in the binding of cAMP in E. coli CRP (4, 26), but these residues are replaced by Leu-144 and Asn-145 in SYCRP1. A check with the Sequence Motif Search (the GenomeNet web server) revealed the cAMP binding motif 1 to be [LIVM]-[VIC]-X(2 amino acid residues)-G-[DENQTA]-X-[GAC]-X(2 amino acid residues)-[LIVMFY](4 amino acid residues)-X(2 amino acid residues)-G and the cAMP binding motif 2 [LIVMF]-G-E-X-[GAS]-[LIVM]-X(amino acid residues 5-11)-R-[STAQ]-A-X-[LIVMA]-X-[STACV]. These motifs are in pairs. SYCRP1 does not match these motifs because of several amino acid substitutions. In motif 1 [VIC] and G (underlined) are replaced by L and H, respectively. In motif 2, A (underlined) is replaced by T (see Fig. 1; E. coli CRP motif 1 is the segment from Leu-30 to Gly-46 and motif 2 is the segment from Ile-71 to Ala-89). Together, these data indicate that SYCRP1 must carry a novel cAMP binding motif. E. coli CRP has a helix-turn-helix motif for DNA-binding in the carboxyl-terminal region (5, 27). From residues Arg-196 to Glu-207 in SYCRP1, this region shows high homology to the latter helix region in E. coli CRP. The specific amino acids that interact with DNA in E. coli CRP are Arg-181, Glu-182, Thr-183, Arg-186, and Lys-189 (5).

SYCRP2 shows high homology (40% similarity) to SYCRP1. However, SYCRP2 lacks several amino acids corresponding to Glu-73, Arg-83, and Ser-84 in E. coli CRP, which participate directly in cAMP binding.

The amino acid sequence of SYPK containing a putative cAMP binding domain is almost the same as that of SYCRP1. SYPK has common cAMP binding motifs; however, the entire predicted amino acid sequence of SYPK comprises 434 amino acids, and the amino- and carboxyl-terminal regions are different from those in E. coli CRP and SYCRP1. As a result of BLAST search (the GenBank^TM BLAST server), it is shown that the cAMP binding site of SYPK is similar to that of the regulatory subunit of PKA in eukaryotes. However, the rest of the SYPK sequence shows less homology to the regulatory subunit of eukaryotic PKA.

Expression and Purification of Recombinant Proteins-- To prepare recombinant proteins encoded by these ORFs for biochemical analysis of the putative cAMP receptor proteins, we constructed expression vectors (pCGA, pHYP, and pPKA) encoding hybrid proteins with His tag or GST as described under "Experimental Procedures." When the entire sypk was cloned into pET-28a, expression of the desired protein was not observed. Therefore, we cloned the ORF into pGEX-2T expression vector (Amersham Pharmacia Biotech) and succeeded in expressing the desired protein as a fusion protein with GST. Unfortunately, the product was insoluble (data not shown). Therefore, the deduced amino acid sequence of the sypk containing a putative cAMP-binding site (193 amino acids; 98-290 amino acids in the entire predicted amino acid sequence, see Fig. 1) was examined. When the partial sypk was cloned into the pGEX-2T expression vector and expressed, a soluble product named GST-pSYPK was obtained. Then, this fusion protein was purified by glutathione-Sepharose 4B column and ion exchange chromatography. Other recombinant proteins (His-tagged SYCRP1 and His-tagged SYCRP2) were purified by Ni²⁺-nitrilotriacetic acid affinity chromatography and ion exchange chromatography. The molecular masses of both His-tagged SYCRP1 (His-SYCRP1) and His-tagged SYCRP2 (His-SYCRP2) were estimated to be 30 kDa by SDS-PAGE analysis, corresponding closely to the theoretical values (Fig. 2). The molecular mass of a major band of GST-pSYPK was estimated to be 45 kDa, but another minor band was also detected at 45.5 kDa, a value that corresponds to the theoretical molecular mass of GST-pSYPK. Probably several amino acids in the carboxyl-terminal region were cleaved without any effect on the binding to cAMP (Fig. 2, lane 6).

View larger version (117K): [in this window] [in a new window]   Fig. 2.   Purification of His-tagged SYCRP1, His-tagged SYCRP2, and GST-pSYPK. SDS-PAGE was carried out using a 12% polyacrylamide gel. The gel was stained with Coomassie Brilliant Blue R-250. Lane 1, cell extract of BL21(DE3)pLysS harboring pCGA after the addition of 1 mM IPTG (15 µg); lane 2, His-tagged SYCRP1 by Ni2+ affinity chromatography and ion exchange chromatography (3 µg); lane 3, cell extract of BL21(DE3)pLysS harboring pHYP after the addition of 1 mM IPTG (15 µg); lane 4, His-tagged SYCRP2 by Ni2+ affinity chromatography and ion exchange chromatography (3 µg); lane 5, cell extract of BL21(DE3)pLysS harboring pPKA after the addition of 1 mM IPTG (15 µg); lane 6, GST-pSYPK by glutathione-Sepharose 4B chromatography and ion exchange chromatography (3 µg). M indicates molecular size markers.

cAMP Binding Activity of Recombinant Proteins-- To determine the cAMP binding ability of each of the expressed proteins, equilibrium dialysis measurements were performed at 25 °C as described under "Experimental Procedures." The results are shown in Fig. 3. Three-fifths of the amount of 5 µM cAMP was bound to 20 µM His-SYCRP1, suggesting that His-SYCRP1 can bind cAMP. His-SYCRP2 showed low cAMP binding activity. We had carried out the equilibrium dialysis measurements using 10 µM cAMP, 10 µM His-SYCRP2, and 10 µM His-tagged galactosidase (Novagen, control sample) as negative control under the same conditions as in Fig. 3 several times (data not shown) and found no cAMP binding activity of His-SYCRP2. Based on these results, we concluded that His-SYCRP2 had no cAMP binding activity. It was noted that very high amounts of cAMP, more than the amounts initially added, were released from 20 µM GST-pSYPK, indicating that GST-pSYPK was purified together with endogenously bound cAMP. To investigate the binding specificity of His-SYCRP1 to cAMP, equilibrium dialysis measurements were performed using 10 µM His-SYCRP1 and 10 µM 5'-AMP or 10 µM cGMP. His-SYCRP1 bound neither 5'-AMP nor cGMP entirely (data not shown); thus, the data indicate that His-SYCRP1 binds cAMP specifically.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Binding activity of cAMP to recombinant proteins determined by equilibrium dialysis. 20 µM His-SYCRP1, 10 µM His-SYCRP2, and 20 µM GST-pSYPK were incubated for 6 h in the presence of 5 µM cAMP at 25 °C. The amounts of bound cAMP were measured by HPLC as described under "Experimental Procedures."

Dissociation Constant (K_d) of His-SYCRP1 from cAMP-- To obtain the K_d value of His-SYCRP1 from cAMP, 0.5-20 µM cAMP was incubated with 5 µM His-SYCRP1 for 6 h at 20 °C or 30 °C for equilibrium dialysis. The K_d value was determined from the revolution lines by least-square analysis on a Scatchard plot (Fig. 4). The K_d value was 2.57 ± 0.08 µM at 20 °C and 2.82 ± 0.09 µM at 30 °C. In this range of temperature, the K_d values were almost constant. These values are physiologically relevant in bacteria because the intracellular cAMP concentration is within 0-10 µM (28). The K_d values of bacterial CRPs are known to be in a order of 10⁵ M in general (29-31). The obtained K_d value for His-SYCRP1 is comparatively lower than that of other bacteria. The bacterial CRPs bind both cAMP and cGMP and the K_d values of both nucleotides are very similar, that is 10⁵ M (29-31). Considering the predicted maximum amounts of bound cAMP on His-SYCRP1, approximately 0.6 molecule of cAMP bound on 1 molecule of His-SYCRP1. This means that a His-SYCRP1 dimer binds 1 molecule of cAMP, as in the case in E. coli CRP (32).

View larger version (86K): [in this window] [in a new window]   Fig. 4.   Determination of the Kd value for His-SYCRP1 by equilibrium dialysis. The binding of cAMP to His-SYCRP1 was measured by equilibrium dialysis at 20 °C () and 30 °C (). 5 µM His-SYCRP1 was incubated for 6 h in the presence of 0.5-20 µM of cAMP at 20 °C or 30 °C, and then the amounts of bound cAMP were measured by HPLC as described under "Experimental Procedures."

Cross-linking Analysis of His-SYCRP1-- To determine whether cyanobacterial CRP forms a dimer, a protein cross-linking experiment of His-SYCRP1 with the zero-length cross-linking reagent EDC was performed. Fig. 5 shows the detecting of a protein-protein cross-linking dimeric His-SYCRP1 band in the absence of cAMP. In E. coli CRP, cAMP has been reported to stabilize the CRP dimer form (33). If cAMP stabilized His-SYCRP1, the proportion of dimeric His-SYCRP1 would be higher when cAMP was added. The addition of cAMP, however, did not increase the proportion of the dimeric His-SYCRP1 band in our preparations (Fig. 5). On the other hand, we could confirm that His-SYCRP1 can bind cAMP molecules as a homodimer. This result suggests that His-SYCRP1 may act as a transcriptional regulator as in the case of other bacterial CRPs.

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Analysis of EDC cross-link product of His-SYCRP1. His-SYCRP1 (4 µg) in the presence or absence of 10 µM cAMP was reacted with 25 mM EDC for 2 h at room temperature as described under "Experimental Procedures." The samples were loaded onto a 10% SDS-polyacrylamide gel for electrophoresis. The gel was stained with Coomassie Brilliant Blue R-250.

Gel Mobility Shift Assay of the Binding of His-SYCRP1 to the E. coli CRP Consensus DNA Sequence-- The carboxyl-terminal region of SYCRP1 shows high homology to the latter part of the helix-turn-helix motif of E. coli CRP that participates in DNA-binding (Fig. 1). Therefore, there is a possibility that SYCRP1 binds some DNA sequences that are similar to E. coli CRP-binding sequences. To clarify this, we performed a gel mobility shift assay using His-SYCRP1 and an E. coli CRP consensus DNA sequence as described under "Experimental Procedures." As shown in Fig. 6A, incubation of His-SYCRP1 with the DNA probe in the presence of cAMP resulted in a retarded band (lanes 2 and 3). The intensity of this band was reduced by the addition of non-labeled DNA probe but not affected by the addition of the same amount of poly(dI-dC) (lanes 4 and 5). This shifted band was not detected in the absence of cAMP (Fig. 6A, lanes 6-9). To examine whether the retarded complex involves His-SYCRP1, Western blotting analysis with tetra-His antibodies (Ni-nitrilotriacetic acid horseradish peroxidase conjugates, Qiagen) was carried out using the gel from the gel mobility shift assay. As shown in Fig. 6B, the Western blotting analysis after incubation of His-SYCRP1 with the DNA probe in the presence of 20 µM cAMP, a His-SYCRP1 protein band was detected at the same position as the shifted band. This band seems arise from a specific interaction of His-SYCRP1 with the DNA probe. His-SYCRP1 alone did not enter the gel in the presence of 20 µM cAMP.

View larger version (47K): [in this window] [in a new window]   Fig. 6.   Gel mobility shift assay with His-SYCRP1 and the consensus sequence for E. coli CRP. A, binding of purified His-SYCRP1 to the consensus sequence DNA for E. coli CRP in the presence (lanes 1-5) or absence (lanes 6-9) of cAMP was examined. The experiments were carried out as described under "Experimental Procedures." The 32P-labeled probe DNA (10,000 cpm, approximately 1 ng) was incubated with the His-SYCRP1, non-labeled DNA, and poly(dI-dC), which were added in the amounts indicated above each lane (lanes 1-5) in the presence of 20 µM cAMP. The 32P-labeled probe DNA (10,000 cpm, approximately 1 ng) and 50 ng of poly(dI-dC) were incubated with the His-SYCRP1 added in the amounts indicated above each lane (lanes 6-9) in the absence of cAMP. The composition of the retarded complex and free DNA are indicated. B, His-SYCRP1 in the retarded complex was detected by Western blot analysis. The non-labeled DNA probe (100 ng) and 50 ng of poly(dI-dC) were incubated with the His-SYCRP1 added in the amounts indicated above each lane (lanes 1-3) in the presence of 20 µM cAMP.

It was concluded that His-SYCRP1 bound to an E. coli CRP consensus DNA sequence, and the binding of His-SYCRP1 to this sequence is dependent on the presence of cAMP in vitro (Fig. 6A). It is unlikely that the His-SYCRP1 protein preparation is contaminated with E. coli CRP, because no band was observed except for His-SYCRP1 on denaturating polyacrylamide gel (Fig. 2), and a band migrating at a position corresponding to the shifted band was observed in Western blotting analysis (Fig. 6B).

SYCRP1 Is a Novel CRP in Cyanobacteria-- Although a sequence-specific interaction between His-SYCRP1 and a DNA sequence was observed in competition experiments (lanes 4 and 5 in Fig. 6A), the identification of the optimum sequence for the binding of His-SYCRP1 remains to be elucidated. The results obtained in the present experiments suggest that SYCRP1 is a novel cAMP receptor protein, which is involved in transcriptional regulation as a mediator of the cAMP signal.

	FOOTNOTES

^* This work was supported by Grant-in-aid 11132208 for general scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences of Japan.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.

^¶ To whom correspondence should be addressed: Dept. of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro, Tokyo 153-8902, Japan. Tel.: 81-3-5454-6631; Fax: 81-3-5454-4333; E-mail: cohmori@mail.ecc.u-tokyo.ac.jp.

	ABBREVIATIONS

The abbreviations used are: CRP, cAMP receptor protein; sycrp1, Synechocystis sp. PCC 6803 cAMP receptor protein-like gene 1; sycrp2, Synechocystis sp. 6803 cAMP receptor protein-like gene 2; sypk, Synechocystis sp. PCC 6803 cAMP-dependent protein kinase-like gene; PKA, cAMP-dependent protein kinase; ORF, open reading frame; GST, glutathione S-transferase; IPTG, isopropyl--D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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