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This cyanobacteria (or blue green alga) is an aquatic prokaryote which is capable o f oxygenic photosynthesis. Purple or green sulphur bacteria are also

photosynthetic but unlike cyanobacteria they do not utilise water as an electron donor. It is generally believed that eukaryotic photosynthetic cells arose from an endosymbiotic event involving an ancient ancestor o f m odem cyanobacteria. Both higher plants and blue green algae show many common characteristics in both the stmcture and mechanism o f the photosynthetic apparatus. In light o f these similarities Synechocystis sp PCC 6803 has been used as a model organism for studying all aspects o f photosynthesis.

Synechocystis sp PCC 6803 has a number o f features which make it

particularly suitable as a model organism. The first o f these is the fact that it can be cultured under a range o f conditions from full photoautotrophy to heterotrophic growth. Photoautotrophic growth can be achieved on a minimal media with no added carbon source in cells with active PSI and PSII complexes. In the presence o f an added carbon source e.g. glucose Synechocystis sp PCC 6803 cells are able to grow in the absence o f PSII. The growth rate o f such cells on supplemented media is not significantly different from that o f photoautotrophic cells. Cyclic electron flow around PSI is sufficient to maintain efficient levels o f growth. Cells which lack PSI grow at a much slower rate independent o f the absence or presence o f PSII. PSI minus mutants can be cultured but are extremely light sensitive because PSII becomes over reduced leading to damage to the photosystem.

The problem o f this light sensitivity can be avoided if the mutants are generated in a strain with a deletion in the apcE gene (Shen et al. 1993). The protein encoded by this gene is responsible for anchoring the phycobilisomes (the light harvesting apparatus o f cyanobacteria) to the photosystems. Synechocystis sp PCC 6803 cells can also be grown in photomixotrophic culture with added glucose or by light activated heterotrophic growth (Anderson & M cIntosh 1991). This form o f heterotrophic growth requires the cells to be exposed to five minutes o f light per day. The wavelength o f light which stimulates this growth, in the range o f 400- 500nm, precludes any involvement o f electron transport involving either photosystem. The authors conclude that the blue light is required as an environmental signal which regulates some aspect o f metabolism or cell division.

There are differences in both the antenna system and the ratio o f the two photosystems between cyanobacteria and algae and higher plants. Cyanobacteria, like red algae, use a phycobilisome system to capture photons and present them to

the photosystems. These are large multi-subunit complexes which are located on the stromal side o f the membrane. The light harvesting portions o f this structure are the phycobiliproteins which are a family o f soluble proteins with covalently bound open-chain tetrapyrroles, known as phycobilins. The precise structure o f the phycobilisomes is able to vary as a method o f adaptation to the environmental conditions.

Another variation between eukaryotic cells and Synechocystis sp PCC 6803 is the ratio o f PSI to PSII. In cyanobacteria I have aheady discussed the differing effects o f disrupting PSI and PSII and this is extended when the relative amount o f each photosystem is examined. Synechocystis cells have approximately six times as many PSI centres as they do PSII. In consequence PSI minus mutants tend to appear more blue in colour in comparison to PSII minus cells which do not differ significantly in appearance to wild type cells. These two features imply that for

Synechocystis sp PCC 6803 cyclic electron flow around PSI is a physiologically

important phenomenon.

There are two further features which make Synechocystis sp PCC 6803 a particularly attractive model organism. Firstly the cells are naturally transformable (reviewed Thiel 1994). The mechanism o f uptake o f DNA has not been discovered. Once inside the cells the transforming DNA undergoes homologous double recombination which replaces the wild type region with the introduced section. A gene conferring antibiotic resistance is usually included on the transforming DNA to provide a method o f selection for transformants. Resistance genes for chloramphenicol, erythromycin, kanamycin and spectinomycin have all been successfully used. An additional selectable marker which can be employed is the

sacB gene (TUid & Collmer 1987, Cai & Wolk 1990). This encodes a levan sucrase and expression in the presence o f sucrose is lethal. If used in combination with a resistance marker, transformants can be selected for then the markers removed. This is achieved by a second transformation with a construct carrying DNA which flanks the original insertion site. Second round transformants (lacking both markers) are selected for in the presence o f sucrose. Spontaneous transformation o f Synechocystis sp PCC 6803 occurs in the range o f 1 in 10^-10^ and depends on the state o f the cells and the length o f the incoming DNA. A final relevant factor in the use o f Synechocystis sp PCC 6803 as a model organism is the

availability o f the entire 3,573,470bp sequence (Kaneko et al. 1996). The presence o f this data, available on a Web page based in Japan, facilitates the construction o f mutants. 3,168 potential protein coding genes have been assigned (Kaneko & Tabata 1997). O f these a function has been deduced by comparison to other known sequences for 1416 but 55% remain unclassified.

Chapter 2