Abstract
The unicellular cyanobacterium Synechococcus sp. PCC 7942 has three psbA genes encoding two different forms of the photosystem II reaction centre protein D1 (D1:1 and D1:2). The level of expression of these psbA genes and the synthesis of D1:1 and D1:2 are strongly regulated under varying light conditions. In order to better understand the regulatory mechanisms underlying these processes, we have constructed a strain of Synechococcus sp. PCC 7942 capable of over-producing psbA mRNA and D1 protein. In this study, we describe the over-expression of D1:1 using a tac-hybrid promoter in front of the psbAI gene in combination with lacI Qrepressor system. Over-production of D1:1 was induced by growing cells for 12 h at 50 μmol photons m-2 s-1 in the presence of 40 or 80 μg/ml IPTG. The amount of psbAI mRNA and that of D1:1 protein in cells grown with IPTG was three times and two times higher, respectively. A higher concentration of IPTG (i.e., 150 μg/ml) did not further increase the production of the psbAI message or D1:1. The over-production of D1:1 caused a decrease in the level of D1:2 synthesised, resulting in most PSII reaction centres containing D1:1. However, the over-production of D1:1 had no effect on the pigment composition (chlorophyll a or phycocyanin/number of cells) or the light-saturated rate of photosynthesis. This and the fact that the total amounts of D1 and D2 proteins were not affected by IPTG suggest that the number of PSII centres within the membranes remained unchanged. From these results, we conclude that expression of psbAI can be regulated by using the tac promoter and lacI Qsystem. However, the accumulation of D1:1 protein into the membrane is regulated by the number of PSII centres.
Similar content being viewed by others
References
Amann E, Brosius J, Ptashne M: Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene 25: 167–178 (1983).
Arnon DI: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1–15 (1949).
Bagdasarian MM, Amann E, Lurz R, Rückert B, Bagdasarian M: Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26: 273–282 (1983).
Bouyoub A, Vernotte C, Astier C: Functional analysis of the two homologous psbA gene copies in Synechocytis PCC 6714 and PCC 6803. Plant Mol Biol 21: 249–258 (1983).
Bradford MM: A rapid sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248–254 (1976).
Bustos SA, Schaefer MR, Golden SS: Different and rapid responses of four cyanobacterial psbA transcripts to changes in light intensity. J Bact 172: 1998–2004 (1990).
Clarke AK, Soitamo A, Gustafsson P, Öquist G: Rapid interchange between two distinct forms of cyanobacterial photosystem II reaction-centre protein D1 in response to photoinhibition. Proc Natl Acad Sci USA 90: 9973–9977 (1993).
Clarke AK, Hurry VM, Gustafsson P, Öquist G: Two functionally distinct forms of the photosystem II reaction-center protein D1 in the cyanobacterium Synechococcus sp. PCC 7942. Proc Natl Acad Sci USA 90: 11985–11989 (1993).
Curtis SE, Haselkorn R: Isolation, sequence and expression of two members of the 32 kDa thylakoid membrane protein gene family from the cyanobacterium Anabaena 7120. Plant Mol Biol 3: 249–258 (1984).
Feinberg AP, Vogelstein B: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Friedberg D, Seijffers J: Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host. Mol Gen Genet 203: 505–510 (1986).
Gingrich JC, Buzby JS, Stirewalt VL, Bryant DA: Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechococcus sp. PCC 7002. Photosynth Res 16: 83–99 (1988).
Golden SS, Bruslan J, Haselkorn R: Expression of a family of psbA genes encoding a photosystem II polypeptide in the cyanobacterium Anacystis nidulans R2. EMBO J 5: 2789–2798 (1986).
Jansson C, Debus RJ, Osiewacz HD, Gurevitz M, McIntosh L: Construction of an obligate phototheterotrophic mutant of the cyanobacterium Synechocystis 6803. Plant Physiol 85: 1021–1025 (1987).
Kulkarni RD, Schaefer MR, Golden SS: Transcriptional and posttranscriptional components of psbA response to high light intensity in Synechococcus sp. strain PCC 7942. J Bact 174: 3755–3781 (1992).
Laemmli UK: Cleave of structural proteins during the assembly of the head of bacteriophage T4. Nature New Biol 277: 680–685 (1970).
Lönneborg A, Lind KL, Kalla SR, Gustafsson P, Öquist G: Acclimation processes in the light-harvesting system of the cyanobacterium Anacystis nidulans following a light shift from white to red light. Plant Physiol 78: 110–114 (1985).
Meade HM, Long SR, Revkun CB, Brown SE, Ausubel FM: Physical and genetic characterization of symbiotic and autotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bact 149: 114–122 (1982).
Miller JH: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1972).
Mulligan B, Schultes N, Chen L, Bogorad L: Nucleotide sequence of a multiple-copy gene for the B protein of photosystem II of a cyanobacterium. Proc Natl Acad Sci USA 81: 2693–2697 (1984).
Pakrasi HB, Vermaas WFJ: Protein engineering of photosystem II. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology, pp. 231–257. Elsevier Science Publishers, Amsterdam (1992).
Prentki P, Krisch HM: In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29: 303–313 (1984).
Price DG, Badger MR: Expression of human carbonic anhydrase in the cyanobacterium Synechococcus PCC 7942 creates a high CO2-requiring phenotype. Plant Physiol 91: 505–513 (1989).
Rao VSK, Brand JJ, Myers J: Cold shock syndrome in Anacystis nidulans. Plant Physiol 59: 965–969 (1977).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).
Schaefer MR, Golden SS: Differential expression of members of a cyanobacterial psbA gene family in response to light. J Bact 171: 3973–3981 (1989).
Schaefer MR, Golden SS: Light availability influences the ratio of two forms of D1 in cyanobacterial thylakoids. J Biol Chem 264: 7412–7417 (1989).
Scherrer K, Darnell JE: Sedimentation characteristics of rapidly labelled RNA from HeLa cells. Biochem Biophys Res Commun 7: 486–490 (1962).
Shapira SK, Chou J, Richaud FV, Casadaban MJ: New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxyl-terminal portion of β-galactoside. Gene 25: 71–82 (1983).
Stark MJR: Multicopy expression vectors carrying the lac repressor gene for regulated high-level expression of genes in Escherichia coli. Gene 51: 255–267 (1987).
Tomioka N, Shinozaki K, Sugiura M: Molecular cloning and characterization of ribosomal RNA genes from a blue-green alga, Anacystis nidulans. Mol Gen Genet 184: 358–363 (1981).
Tyystjärvi T, Aro E-M, Jansson C, Mäenpää P: Changes in amino acid sequence in pest-like area and QEEET-motif affect degradation rate of D1 polypeptide in photosystem II. Plant Mol Biol, in press (1994).
Van der Plas J, Hegeman H, de Vrieze G, Tuyl M, Borias M, Weisbeek P: Genomic integration system based on pBR 322 sequences for the cyanobacterium Synechococcus sp. PCC 7942: transfer of genes encoding plastocyanin and ferrodoxin. Gene 95: 39–48 (1990).
Vermaas WFJ, Ikeuchi M: Photosystem II. In: Bogorad L, Vasil IK (eds) The Photosynthetic Apparatus: Molecular Biology and Operation. Cell Culture and Somatic Cell Genetics of Plants, vol. 7B, pp. 26–85. Academic Press, San Diego, CA (1991).
Viera J, Messing J: The pUC plasmids, a M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268 (1982).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Soitamo, A.J., Zhou, G., Clarke, A.K. et al. Over-production of the D1 protein of photosystem II reaction centre in the cyanobacterium Synechococcus sp. PCC 7942. Plant Mol Biol 26, 709–721 (1994). https://doi.org/10.1007/BF00013756
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00013756