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Photorespiratory 2-phosphoglycolate (2PG) metabolism is essential for photosynthesis in higher plants but thought to be superfluous in cyanobacteria because of their ability to concentrate CO₂ internally and thereby inhibit photorespiration. Here, we show that 3 routes for 2PG metabolism are present in the model cyanobacterium Synechocystis sp. strain PCC 6803. In addition to the photorespiratory C2 cycle characterized in plants, this cyanobacterium also possesses the bacterial glycerate pathway and is able to completely decarboxylate glyoxylate via oxalate. A triple mutant with defects in all 3 routes of 2PG metabolism exhibited a high-CO₂-requiring (HCR) phenotype. All these catabolic routes start with glyoxylate, which can be synthesized by 2 different forms of glycolate dehydrogenase (GlcD). Mutants defective in one or both GlcD proteins accumulated glycolate under high CO₂ level and the double mutant …