| Myxoxanthophyll is a glycosylated carotenoid that is common in cyanobacteria but whose function and biosynthetic pathway remain unresolved. In this dissertation, four unknown open reading frames (sll1213, slr1293, slr1125 and sll0254) in the genome of the cyanobacterium Synechocystis sp. PCC 6803 were investigated to identify enzymes and intermediates in the myxoxanthophyll biosynthesis pathway and to probe the biological significance of myxoxanthophyll. The sll1213 open reading frame putatively encoding fucose synthetase was deleted and a pleiotropic phenotype was obtained; cells lost myxoxanthophyll but new carotenoid glycosides emerged. Moreover, the mutant showed a very thin glycocalyx (S-layer) and peptidoglycan layer, both of which are components of the cell wall. This apparently led to cell aggregation in liquid as well as on plates. In addition, this mutant showed significant accumulation of cyanophycin and a remarkable disorganization of thylakoid membranes. The slr1293 open reading frame was identified by genomic similarity searching for C-3,4 desaturase gene orthologues. Results of expression of Slr1293 in E. coli strains accumulating neurosporene or lycopene and deletion of slr1293 in Synechocystis confirm such a C-3,4 carotenoid desaturase function. The slr1125 open reading frame, hypothesized to encode a glycosyl transferase based on homology studies, was deleted. In the resulting mutant, a pool of carotenoid precursors accumulated and were identified as potential intermediates in the carotenoid biosynthesis pathway. Based on these results, neurosporene is a primary candidate to be the branch point molecule between carotene and myxoxanthophyll biosynthesis in this cyanobacterium. Full deletion of the sll0254 open reading frame could not be obtained but partial segregants lacked photosytem I. Cyclized carotenoids in wild type were replaced by linear carotenoid glycosides, with 9-13 conjugated double bonds. These results provide preliminary evidence that Sll0254 is the lycopene cyclase or a component thereof. On the basis of this study, myxoxanthophyll turns out to be a critical factor in the physiology and structure of this cyanobacterium. Reducing myxoxanthophyll levels led to thylakoid membrane disorganization, cell wall deformation, loss of the S-layer and increase in cyanophycin. Reasons for each of these effects are discussed, but it is clear that myxoxanthophyll is an important component of both thylakoids and the cell wall, and that this unusual compound is a major player in the properties of cyanobacterial membranes. |
