A Phycocyanin-Deficient Mutant of Synechocystis PCC 6714 with a Single-Base Substitution Upstream of the cpc Operon

Abstract

The structure and expression of the cpc operon encoding phycocyanin subunits and linker polypeptides in a phycocyanin-deficient mutant (PD-1) and the wild-type of Synechocystis PCC 6714 were analyzed. The results of sequence and Northern blot analyses of the wild type indicate that the cpc operon consists of cpcB, cpcA, cpcC1, cpcC2 and cpcD, in that order. The levels of the transcripts in PD-1 were one-tenth to one-sixth as high as those in the wild type. In the PD-1 genome, a single-base substitution of C for T has occurred at base 259 upstream of the translational initiation codon of cpcB (at three bases downstream of the putative -10 region). To evaluate the in vivo transcription activities of these promoters in a cyanobacterium, we constructed vectors for the transformation of Synechococcus PCC7942, pANY1 and pANY2, which contain the upstream region of cpcB of the wild type (pANY1) or PD-1 (pANY2) and the promoter-less luxAB fusion. The bioluminescence of the transformants with pANY2 was one-tenth to one-sixth as high as that with pANY1. The coincidence of the results of Northern analysis and the promoter assay shows that the phycocyanin deficiency of PD-1 is due to the single-base substitution in the upstream region of the cpc operon.

Thymine at —5 Is Crucial for cpc Promoter Activity of Synechocystis sp. Strain PCC 6714

“…The wild type and PD-1 mutant of Synechocystis sp. strain PCC 6714 were grown as described previously (13,14) for use for RNA extraction. The wild type and transformants of Synechococcus sp.…”

“…strain PCC 6714, PD-1 (13). The cpc operon comprises cpcBAC1C2D, which encodes phycocyanin and linker polypeptides located in peripheral rods in principal cyanobacterial light-harvesting antennae, i.e., phycobilisomes.…”

“…We previously reported that substitution of C for T at 259 bp upstream of the cpcB initiation codon of Synechocystis sp. strain PCC 6714 decreased the levels of transcripts drastically (13). To determine whether or not the site of the substitution is upstream of the transcription initiation site, transcripts of the cpc operon from Synechocystis sp.…”

“…1A). Total RNA (10 to 20 g) of the strains, which was prepared by the hot-phenol method described previously (13), was annealed with about 2 pmol of an end-labeled oligonucleotide, PE1 (5Ј-ATGGCTGCTCTCC ATAAAAC-3Ј) (18) and then extended with a Moloney murine leukemia virus reverse transcriptase, ReverTra Ace (Toyobo, Osaka, Japan), for 30 min at 50°C. The reaction was stopped with formamide loading buffer.…”

“…To examine the effect of a nucleotide substitution at Ϫ5 on the transcription level, cpc promoter regions with A and G at Ϫ5, respectively, were prepared. In addition, promoter regions with T (wild type) and C (PD-1 mutant), which were constructed previously (13), and the promoter-luxAB fusions, were used for reporter assaying in a cyanobacterium. It has been shown that the mutational effect of the cpc promoter region of Synechocystis sp.…”