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Journal of Bacteriology, November 2003, p . 6477-6480, Vol . 185, No . 21

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

Masahiko Imashimizu,1 Shoko Fujiwara,1,2 Ryohei Tanigawa,3 Kan Tanaka,3 Takatsugu Hirokawa,4 Yuji Nakajima,5 Junichi Higo,1 and Mikio Tsuzuki1,2*

School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji 192-0392,1 CREST, Japan,2 Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032,3 Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology, Koutou-ku, Tokyo 135-0064,4 Advanced Technology Research Center, Mitsubishi Heavy Industries, Ltd., Kanazawa-ku, Yokohama 236-8515, Japan5

Received 1 May 2003/ Accepted 3 August 2003


   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 .


   TEXT

 
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 -5 position, and we demonstrated that T at the -5 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-2 s-1 . 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 µg) 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 (5'-ATGGCTGCTCTCCATAAAAC-3') (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 5' end of the cpc mRNA was found to be located 254 bp upstream of the cpcB initiation codon in both the wild type and PD-1 . Figure 1B shows that a substitution occurred at 5 bp upstream of the transcription initiation site (-5) . The transcription initiation sites of the cpc operons in seven species of cyanobacteria have been determined so far . The -5 position of the promoter in Synechocystis sp . strain PCC 6714 is located in the nonconserved region between the -10 element and +1 . However, T at 4 bp downstream of the 3' end of a putative -10 element, GTATAA, seems to be conserved in unicellular cyanobacteria except for Synechocystis sp . strain PCC 9413 .


FIG . 1 . The promoter region of the cpc operon . (A) Primer extension analysis of cpc operon transcripts . Lanes 1 and 2, reactions with mRNA isolated from Synechocystis sp . strain PCC 6714, the wild type and PD-1 mutant, respectively . The position of the 5' end of the transcripts, indicated by a gray box, was determined by comparing the migration of a DNA sequence ladder extended with the same primer as that used for primer extension, i.e., PE1 . (B) Sequence alignment of the promoter region of the cpc operon . The sequences of the cpc operon promoter regions of Synechocystis sp . strain PCC 6714 (this work), Synechocystis sp . strain PCC 6701 (1), Synechocystis sp . strain PCC 9413 (15), Synechococcus sp . strain PCC 7942 (10), Synechococcus sp . strain PCC 7002 (6), Anabaena sp . strain PCC 7120 (3), and Calothrix sp . strain PCC 7601 (4) were aligned with CLUSTAL W (23) . The transcriptional start sites (+1) are indicated by boxes . Perfectly conserved nucleotides are indicated by shading . The substitution site of the PD-1 mutant at -5 is indicated by an arrow.

 
In vivo promoter activity in Synechococcus sp . strain PCC 7942. To examine the effect of a nucleotide substitution at -5 on the transcription level, cpc promoter regions with A and G at -5, 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 . strain PCC 6714 is also reflected in Synechococcus sp . strain PCC 7942, regardless of their somewhat different genetic backgrounds (13) . Therefore, we performed reporter assays on Synechococcus sp . strain PCC 7942 by using an available plasmid, pAM1414 (2) . The promoter regions (763 bp, -511 to +252 [when the transcription initiation site is +1]) of the wild type and PD-1 were inserted in a region upstream of promoterless luxAB genes in pAM1414 (2), and then -5T of the wild-type promoter was changed to A and G, respectively, by site-directed mutagenesis . The primers used were MF1 (5'-ACTAAGCTGATCCGGTGGAT-3'), MR1 (5'-GTGGCTGATAAGTGAGAAGG-3'), MUT (5'-CGAGTGATCCATTAATCTCC-3'), R1A (5'-GTAAACTGTGGGATTGCAAA-3'), and R1G (5'-GTGAACTGTGGGATTGCAAA-3'), according to the method of Ito et al . (8) . Synechococcus sp . strain PCC 7942 was transformed with the four kinds of plasmids carrying the cpc promoters of Synechocystis sp . strain PCC 6714, with T, C, A, and G at -5, respectively . The promoter activities of the transformants were determined from the in vivo bioluminescence of luxAB gene products according to the method of Maeda et al . (11) . One hundred microliters of a cell culture at the exponential-growth phase, which was diluted to about 1 µg of chlorophyll/ml, was transferred to a test tube and then mixed with 2 µl of a 0.1% n-decanal emulsion . The bioluminescence of the cell suspension was determined as described previously (13) . As shown in Fig . 2, any substitution for T at the -5 position reduced the intrinsic promoter activity .


FIG . 2 . In vivo promoter activity in Synechococcus sp . strain PCC 7942 . The activity was measured as the expression of luxAB reporter genes . The nucleotide indicated at the bottom of each column refers to the nucleotide located at -5 in each promoter . The data are expressed as means plus the standard deviations of the values for three independent experiments.

 
In vitro promoter activity with Synechococcus sp . strain PCC 7942 RNAP. To determine whether or not the effect of the nucleotide at -5 on the transcription level is mediated by a transcription factor, we performed in vitro transcription with purified Synechococcus sp . strain PCC 7942 RNA polymerase (RNAP) (Fig . 3) . The holoenzyme was reconstituted by mixing the core RNAP with recombinant sigma factor RpoD1 . The RNAP core enzyme and principal sigma factor (RpoD1) were purified according to the previously described methods of Goto-Seki et al . (7) . The core enzyme was mixed with a threefold molar excess of purified sigma protein (RpoD1), which was followed by incubation for 30 min at 30°C to allow formation of the holoenzyme . Single-round transcription reactions were performed under standard conditions for the E . coli RNAP with modifications (16) . A transcription reaction mixture (35 µl) comprising 0.1 pmol of template DNA and 3 pmol of RNAP in T buffer (22) was incubated for 20 min at 30°C, after which RNA synthesis was initiated by the addition of 15 µl of a prewarmed substrate mixture containing 160 µM concentrations each of ATP, GTP, and CTP, as well as 50 µM UTP and 2 µCi of [{alpha}-32P]UTP (Amersham Biosciences Co., Uppsala, Sweden) in T buffer . After incubation for 5 min at 30°C, the reaction was terminated by the addition of 50 µl of an ice-cold stop solution containing 40 mM EDTA and E . coli tRNA (300 µg/ml), and then the nucleic acids were precipitated with ethanol . The transcripts were electrophoresed through a 5% polyacrylamide gel containing 8 M urea and then examined with a BAS1000 image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan) . The lengths of transcripts were estimated with reference to the lengths of known transcripts of E . coli RNAP . The experiments were performed at least twice to confirm the reproducibility . The template for each reaction was prepared as follows: a 296-bp fragment comprising -205 to +91 of the cpc promoter with T, C, A, or G was PCR amplified with primers, i.e., ITB (5'-GTTCCCATTGAACATCAAGG-3') and ITC (5'-CAACCCAAGGGAAAGTTACA-3') . A 326-bp fragment comprising -205 to +121 of the cpc promoter of pANY1 (13), which was used as an internal control for each reaction, was PCR amplified with primers, i.e., ITB and ITD (5'-AAGGGAATTTATGAGAGGCG-3') . As a result, 91- and 121-nt transcripts were produced through the respective transcription reactions, and so the expected promoter recognition could be detected . The in vitro promoter activity with the four kinds of promoters showed a similar tendency to the in vivo promoter activity in Synechococcus sp . strain PCC 7942 (Fig . 2 and 3) .


FIG . 3 . In vitro transcription experiments on reconstituted RNAP of Synechococcus sp . strain PCC 7942 . (A) DNA templates carrying the cpc promoters with different nucleotides at -5 were examined by single-round transcription assaying in vitro . The nucleotide indicated at the bottom of each lane refers to the nucleotide located at -5 in each template . The target 91-nt and control 121-nt transcripts are indicated by arrows . (B) The relative band intensity for each promoter was quantified . The quantification was performed by dividing the band intensity of each 91-nt transcript with that of each 121-nt control transcript given in panel A . Each quantity represents the average of two independent experiments.

 
In vivo promoter activity in E . coli. Cyanobacterial RNAPs are known to have characteristic subunit compositions, which are not found for other eubacterial RNAPs (19, 20) . To determine whether the effect of -5T in the cpc promoter of Synechocystis sp . strain PCC 6714 is cyanobacterial RNAP specific or not, we used E . coli as a host for the in vivo promoter assay . The promoters (458 bp, -206 to +252) with T, C, A, and G at -5, respectively, were inserted into the BamHI site upstream of the lacZ gene in the promoterless lac operon fusion vector pRS415 (21) . The E . coli JM109 transformants with the plasmids were grown at 37°C in M9 medium (12) containing 0.5% Casamino Acids and thiamine (100 µg/ml) until an optical density at 600 nm of approximately 0.8 was achieved . ß-galactosidase activity was measured as described previously (12) . We confirmed that transcription from the plasmid backbone accounted for less than 3% of all promoter activity . As shown in Fig . 4, the tendency of the four types of promoter activity in E . coli was different from that in Synechococcus sp . strain PCC 7942 (Fig . 2), i.e., the promoter activities in E . coli exhibited no -5 nucleotide specificity .


FIG . 4 . In vivo promoter activity of E . coli. The activity was measured as the expression of the lacZ reporter gene . The nucleotide indicated at the bottom of each column refers to the nucleotide located at -5 in each promoter . The rightmost column, designated as -P, shows the activity with a promoterless vector used as a control . The data are expressed as means plus the standard deviations of the values for three independent experiments.

 
Comparison of the RNAP structure between cyanobacteria and E . coli. On the assumption that a potential factor is not copurified with RNAP, the results of in vitro transcription in Synechococcus sp . strain PCC 7942 indicate that the -5T recognition is not mediated by a transcription factor but may be directly performed by RNAP . In E . coli, however, an effect of substitution of C for T at -5 on the promoter activity could not be observed in vivo . This suggests that the requirement for T at the -5 position appears to be specific for a cyanobacterial RNAP-promoter combination .

Cyanobacterial RNAP shows two remarkable structural differences in the ß' subunit from that of E . coli (Fig . 5): One is a split separating ß' from {gamma} (19, 20), and the other is a large insertion, which possibly interacts with DNA, as in the jaw module of the Rpb1 subunit of yeast RNAP II (5) . It is conceivable that the effect of T at position -5 is somehow related to the presence of the split separating ß' from {gamma} and/or the large insertion in the C-terminal region of ß' in cyanobacterial RNAP .


FIG . 5 . Schematic sequence comparison of the RNAP ß' subunits of E . coli (top) and cyanobacterium Synechocystis sp . strain PCC 6803 (bottom) . The boxes designated A through H indicate evolutionally conserved regions (9) . Striped boxes indicate the hypervariable regions (24); in the case of cyanobacteria, this region represents the insertion domain . The split site of the cyanobacterial subunit is indicated by an arrow . The N-terminal portion comprising regions A through D is the {gamma} subunit, and the C-terminal portion comprising regions E through H is the ß' subunit . The gradations on the scale bar at the bottom each indicate 100 amino acids . The primary sequences of ß'{gamma} cyanobacterial subunits, which were available in genomic databases, were very similar to each other (data not shown) . Therefore, the sequences of Synechocystis sp . strain PCC 6803, the species most related to both Synechocystis sp . strain PCC 6714 and Synechococcus sp . strain PCC 7942, were utilized as cyanobacterial representatives . The amino acid sequences of E . coli and Synechocystis sp . strain PCC 6803 were obtained through GenBank (http://www.ncbi.nlm.nih.gov/Entrez/) and CyanoBase (http://www.kazusa.or.jp/cyano/), respectively.

 


   ACKNOWLEDGMENTS

 
We thank K . Okada, Y . Maru, and K . Ikeda of Tokyo University of Pharmacy and Life Science, and we also thank T . Fujisawa and members of M . Ohmori's lab . at the University of Tokyo for helpful discussions . We are also grateful to S . S . Golden of Texas A&M University for the plasmid pAM1414 and to K . Sugimoto of Tokyo University of Pharmacy and Life Science for support in the experimental work .

This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Tokyo, Japan (grant nos . 13640657, 13740463, and 13874112) and the Promotion and Mutual Aid Corporation for Private Schools .


   FOOTNOTES

 
* Corresponding author . Mailing address: School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji 192-0392, Japan . Phone: 81-426-76-6713 . Fax: 81-426-76-6721 . E-mail: mtsu{at}ls.toyaku.ac.jp .


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