Mobility of pigment-protein complexes in cyanobacteria
Phycobilisomes, the light harvesting complexes of cyanobacteria are highly mobile, fluorescent complexes known to diffuse freely on the thylakoid membranes, interacting with the reaction centre complexes to mediate efficient photosynthesis. The primary aim of this project is to establish what processes require this rapid movement of the complexes using a number of genetic, biochemical and microscopic techniques. The cyanobacterial species used extensively in the work presented in this thesis are the fully sequenced, naturally transformable Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942. The latter lends itself particularly well to quantitatively elucidating the diffusion rate of fluorescent complexes, but qualitative detection of mobile fluorescent complex is also feasible with Synechocystis 6803. State transitions are observed in cyanobacteria upon the alteration of illumination conditions. A rapid redistribution of excitation energy between the reaction centres is observed. This was investigated using high osmotic strength buffers to fix phycobilisomes to reaction centres they were associating with upon their addition, thus inhibiting their mobility, as adjudged by spectroscopy and microscopy using the Fluorescence Recovery after Photobleaching (FRAP) technique. It was found that mobile phycobilisomes are required for cells to be capable of state transitions. Non-Photochemical Quenching (NPQ) is a protective mechanism seen in iron- deprived cyanobacteria. Extensively studied in plants, its supposed function is to dissipate excess energy as heat to prevent photodamage to the reaction centres. Using Synechocystis 6803 and the techniques described above, phycobilisome mobility was determined to be critical to NPQ induction, and the interaction with IsiA in cyanobacteria was proposed as being involved in the process. A previously inactivated gene thought to be involved in state transitions, rpaC, was over-expressed in Synechocystis 6803 and knocked out in Synechococcus 7942 and gave pleiotropic effects. The conclusion that the binding of phycobilisomes to PSII is predictably stronger than to PSI was exploited by comparing the strength of the binding in the Synechococcus 7942 mutant with the wild type. Data were suggestive of the protein being involved in phycobilisome to PSII binding. Psb28* mutants of both species used in this thesis were extensively characterised, as the cells also presented a highly unusual mobile PSII phenotype. Psb28 is possibly involved in maintaining thylakoid membrane organisation.