Synechocystis sp. PCC6803 is a strain of unicellular, freshwater cyanobacteria. Synechocystis sp. PCC6803 is capable of both phototrophic growth by oxygenic photosynthesis during light periods and heterotrophic growth by glycolysis and oxidative phosphorylation during dark periods. Gene expression is regulated by a circadian clock and the organism can effectively anticipate transitions between the light and dark phases.
Evolutionary history Cyanobacteria are photosynthetic prokaryotes that have existed on Earth for an estimated 2.7 billion years. The ability of cyanobacteria to produce oxygen initiated the transition from a planet consisting of high levels of carbon dioxide and little oxygen, to what has been called the Great Oxygenation Event where large amounts of oxygen gas were produced. Cyanobacteria have colonized a wide diversity of habitats, including fresh and salt water ecosystems, and most land environments. Phylogenetically, Synechocystis branches off later in the cyanobacterial evolutionary tree, further from the ancestral root (Gloeobacter violaceus). Synechocystis, which is non-diazotrophic, is related to other model cyanobacteria that can fix nitrogen. Thus, it has been proposed that Synechocystis originally possessed the ability to fix nitrogen gas into ammonia, but lost the genes required for a fully functioning nitrogen fixation (nif) gene cluster.
Growth and use as a model organism Cyanobacteria are model microorganisms for the study of photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Synechocystis sp. PCC6803 is one of the most highly studied types of cyanobacteria as it can grow both autotrophically or heterotrophically in the absence of light. It was isolated from a freshwater lake in 1968 and grows best between 32 and 38 degrees Celsius. Synechocystis sp. PCC6803 can readily take up exogenous DNA, in addition to up taking DNA via electroporation, ultrasonic transformation and conjugation. The photosynthetic apparatus is very similar to the one found in land plants. The organism also exhibits phototactic movement. Synechocystis sp. PCC6803 can be grown on either agar plates or in liquid culture. The most widely used culture medium is a BG-11 salt solution. The ideal pH is between 7 and 8.5. A light intensity of 50 μmol photons m−2 s−1 leads to best growth. Bubbling with carbon dioxide enriched air (1–2% CO2) can increase the growth rate, but may require additional buffer to maintain pH Selection is typically performed by antibiotic resistance genes. Heidorn et al. 2011 experimentally determined in Synechocystis sp. PCC6803 the ideal concentrations of kanamycin, spectinomycin, streptomycin, chloramphenicol, erythromycin, and gentamicin. Cultures can be kept on agar plates for approximately 2 weeks and re-streaked indefinitely. For long term storage, liquid cell cultures should be stored in a 15% glycerol solution at -80 degrees Celsius.
Genome The genome of Synechocystis sp. PCC6803 is contained within approximately 12 copies of a single chromosome (3.57 megabases), three small plasmids: pCC5.2 (5.2 kb) pCA2.4 (2.4 kb), and pCB2.4 (2.4 kb) and four large plasmids: pSYSM (120 kb), pSYSX (106 kb), pSYSA (103kb), and pSYSG (44 kb). The genome of Synechocystis sp. PCC6803 is the fourth genome to be completely sequenced, and the first phototrophic organism to have its genome fully sequenced.
Additional strains The primary strain of Synechocystis sp. is PCC6803. Further modifications of the parent PCC6803 strain have been created, such as a sub-strain lacking photosystem 1 (PSI). The other widely used sub-strain of Synechocystis sp. is a glucose tolerant strain, ATCC 27184. The parent PCC 6803 strain cannot utilize external glucose.
Light-activated heterotrophy Synechocystis sp. PCC6803, sub-strain ATCC 27184 can live heterotrophically in the dark on the carbon source glucose, but for yet unknown reasons requires a minimum of 5 to 15 minutes (blue) light per day. This regulatory role of light is intact in both PSI and PSII deficient strains. Some glycolytic genes are regulated by the gene sll1330 under light and glucose-supplemented conditions. One of the most important glycolytic genes is fructose-1,6-bisphosphate aldolase (fbaA). The mRNA level of fbaA is increased under light and glucose-supplemented conditions.
Native CRISPR-Cas system The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats – CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. Synechocystis sp. PCC6803 contains three different CRISPR-Cas systems: type I-D, and two versions of type III. All three CRISPR-Cas systems are localize on the pSYSA plasmid. All cyanobacteria are lacking the type II system, which has been widely adapted for genetic engineering purposes across many species.
RNA polymerase and sigma factors RNA polymerase (RNAP) and sigma factors are necessary proteins for transcription of DNA into messenger RNA (mRNA). Eubacterial RNAP holoenzymes consist of a core with four major subunits α2 ββ'. In cyanobacteria, β' is formed from two smaller subunits (у and β'), which corresponds to RNAPs in plant chloroplasts. The beta subunits are responsible for binding the RNAP to the DNA, preventing premature dissociation. In Escherichia coli, the beta "clamp" first binds loosely and tightens as the RNAP approaches the start codon (AUG). In cyanobacteria, the beta clamp binds tightly at initial binding. The effect of this difference is that synthetic repressible promoters do not function as expected in Synechocystis sp. PCC6803. In E. coli, a repressor binds the DNA operon and dislodges RNAP due to the loosely bound beta clamp, whereas in Synechocystis, the RNAP is tightly bound leading the reverse phenomenon where the repressor is knocked off the DNA. Thus the gene is not effectively repressed. Synechocystis possesses the 70S sigma factor (σ70), which can be divided into three groups. Group 1 sigma factors are critical for cell viability. Group 2, similar in structure to Group 1, is not essential for cell vitality. Group 3 is structurally different and involved with survival under stress conditions. Synechocystis sp. PCC6803 lacks the σN factor found in other organisms, such as Escherichia coli, which is involved with transcribing genes related to nitrogen, but is nonetheless able to metabolize nitrogen.
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