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

Abstract

The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp. strain PCC 6714. This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon. Any substitution for T at the ؊5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp. strain PCC 7942 but not the in vivo activity in Escherichia coli. This suggests that the requirement of ؊5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination.Cyanobacteria are photosynthetic prokaryotes that are considered to be the ancestors of chloroplasts. Cyanobacteria have been investigated intensively, as a model of oxygen-evolving photosynthetic organisms, in various aspects such as photosynthesis and gene expression.We have found that a single base substitution has occurred in the cpc operon in a phycocyanin-deficient mutant of the cyanobacterium Synechocystis 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. In this study, we found that the substitution site that drastically reduces the transcription level is the Ϫ5 position, and we demonstrated that T at the Ϫ5 position is crucial for the promoter activities determined with in vivo and in vitro systems of Synechococcus sp. strain PCC 7942.Organisms and growth conditions. 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 7942 were grown at 30°C in BG11 medium (17) with aeration with ordinary air under continuous illumination at 30 microeinsteins m Ϫ2 s Ϫ1 . Escherichia coli JM109, as the host for plasmid propagation, was grown in Luria-Bertani medium at 37°C.Determination of the transcription initiation site of the cpc operon. 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. strain PCC 6714, the wild type and the PD-1 mutant, were analyzed by means of primer extension (Fig. 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. The products were electrophoresed on a 6% polyacrylamide gel along with a sequencing ladder. The 5Ј end of the cpc mRNA was found to be l...

Fatty acid production in genetically modified cyanobacteria

“…2). The P cpc promoter of the cpc operon, which encodes the photosynthesis antenna protein phycocyanin (26), and the P rbc promoter of the rbc operon, which encodes ribulose 1,5-bisphosphate carboxylase (27), both drive highly expressed genes in SD100. In SD223 and SD225, two poly-3-hydroxybutyrate (PHB) synthesis genes (slr1993 and slr1994) were deleted.…”

Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity

“…S3). Therefore, the same nucleotide substitution and an additional T→G substitution, which has also been shown to lower transcription (Imashimizu et al, 2003), were introduced into the wild type and the DCpcC2 and DCpcC1C2 mutants via two-step homologous recombination. The promoter region around the T nucleotide was first replaced with an npt1/ sacRB cassette.…”

“…A Synechocystis sp. PCC 6714 mutant with a T→C substitution 5 bp downstream of the transcription initiation site of the cpcBAC1C2D operon (PD-1; Imashimizu et al, 2003) and containing only one-third the level of PC (Nakajima et al, 1998) demonstrated reduced photoinhibition and 20% to 30% higher photosynthesis, as determined by higher oxygen evolution rates, in dense cultures under high light (Nakajima and Ueda, 1997). However, biomass accumulation and growth were not quantified in these studies.…”

Phycobilisome rod mutants in Synechocystis sp. strain PCC6803

“…The site was mapped 251 bp upstream from the translation start for cpcB in Synechocystis sp. strain PCC6714 (Imashimizu et al, 2003), a strain that is closely related to Synechocystis sp. strain PCC6803.…”