Strains and Growth Conditions. Anabaena sp. PCC 7120 (Anabaena 7120 from hereon), its derivative strains, and Anabaena variabilis were grown in BG11 or BG11₀ media (1). Escherichia coli was grown in LB medium at 37°C. The strain DH5a was used for all general cloning purposes. The strain BL21(DE3) was used for protein overproduction (2).

Isolation of Ca²⁺-Binding Protein. Anabaena 7120 cells in buffer A (20 mM Tris·Cl, pH 7.8/2 mM CaCl₂/20 mM NaCl) were broken with a French press at 20,000 psi. The extracts were centrifuged at 18,000 × g for 40 min at 4°C. To the supernatant, (NH₄)₂SO₄ was added to 60% saturation, and the solution was stirred for 30 min before being centrifuged as above. The pellet was discarded. To the supernatant, (NH₄)₂SO₄ was added to 75% saturation. The solution was stirred and centrifuged as above. The pellet was resuspended in 20 ml of buffer A. The proteins were then loaded on a DEAE-cellulose column (2 × 25 cm) and eluted with a NaCl gradient from 20 to 800 mM in a total volume of 800 ml in buffer A. The fractions containing Ca²⁺-binding proteins were pooled and concentrated by ultrafiltration before being loaded onto a Sephadex G-50 column (2 × 40 cm). The column was eluted with buffer A at a flow rate of 15 ml·h^-1. The Ca²⁺-binding protein was separated by SDS/PAGE before being transferred to a poly(vinylidene difluoride) (PVDF) membrane and sequenced. The determined sequence was used to search the genome database of Anabaena 7120 (www.kazusa.or.jp/cyano). An ORF (alr1010) was found to encode a protein whose N-terminal sequence matched exactly the sequence of the isolated Ca²⁺-binding protein. The ORF was named ccbP (GenBank accession no. AY919604).

DNA Manipulation and Protein Overproduction. Isolation of total cyanobacterial genomic DNA was carried out as described by Zhao et al. (3). For Southern hybridization, total DNA was digested with XbaI and separated on an agarose gel before transfer to nitrocellulose membrane and hybridization with a radioactive probe for ccbP. For construction of the CCBP-M strain of Anabaena 7120, ccbP with its flanking regions was amplified by PCR with primers 5'-TTGGATCCCTGGTGCGTTCACAGCAC-3' (BamHI) and 5'-TTGGATCCGCTTAATATACAGTTTG-3' (BamHI). The resulting fragment was cloned into pGEM-T, and the gene npt encoding neomycin resistance was inserted into the unique BalI site of ccbP. The interrupted ccbP gene was cloned into pRL277 (4) for ccbP inactivation. Transformation of Anabaena 7120 and A. variabilis was carried out by conjugal transfer (4). The plasmid for copper-induced expression of ccbP in Anabaena 7120 (pPpetE-ccbP) was constructed essentially as described (5), except that pRL25C was used as the vector. The plasmid for copper-induced expression of a rat calmodulin (CaM) gene cam (GenBank accession no. XP237641) was constructed as follows. The plasmid pSE380CaM, which contained the rat cam, was digested with NcoI and BamHI. The generated fragment containing the rat cam was used to replace ccbP in pPpetE-ccbP to generate pPpetE-CaM. When needed, a gene encoding erythromycin resistance (ery^R) was inserted into the npt gene of the pRL25C derivative plasmids. The plasmids were then transformed into Anabaena 7120. Induction of gene expression was carried out by adding 2.5 m M CuCl₂ to the medium as described (5). The construction of a plasmid for studying spatial expression of ccbP by using GFP was performed as follows. The 0.8-kb fragment upstream of ccbP was amplified by PCR with primers 5'-TTAGATCTGGTGCGTTCACAGCAC-3' (BglII) and 5'-TTCTAGACTGCTTAATATACAGTTTG-3' (XbaI). The amplified fragment was cloned into pET-3d (Novagen) digested with the same enzymes. The modified GFP gene (6) was then inserted into the NcoI site of the plasmid. The fragment containing P_ccbP-gfp was cloned into pRL25C to generate pPccbP-gfp.

For overproduction of recombinant CcbP, the ccbP gene was amplified by PCR with primers 5'-TTCCATGGCCAGTGTAGAACGCG-3' (NcoI) and 5'-TTGGATCCTGCTTAATATACAGTTTG-3' (BamHI). The amplified fragment was cloned into pET-3d, and ccbP was expressed as described (7). Overproduced rCcbP was purified as native CcbP and verified by N-terminal sequencing.

The gene for obelin (obe) from Obelia geniculata (8) was used as a [Ca²⁺]_i indicator in Anabaena 7120. First, a tac promoter was amplified by PCR with primers 5'-AAGCAGATCTCAAGGCGCACTCCCGTTCTG-3' (BglII) and 5'-TTGCGGATCCTGTGTGAAATTGTTATCCG-3' (BamHI) using pGX4T1 (Pharmacia) as template. The amplified fragment was digested with BglII and BamHI and cloned into pAM505 (6) digested by BamHI to generate pAM505tac. The promoter direction was the same as that of the npt gene on pAM505. The obe gene was amplified by PCR from the cDNA with the primers 5'- AAGAGAGCTCATGGCTTCCAAATACGC-3' (SacI) and 5'- AAAAGTCGACTTAGGGGACTCCATTTC-3' (SalI). The amplified fragment was digested with SacI and SalI and cloned into pAM505tac to generate pAM505-ob.

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