In cyanobacteria many compounds, including chlorophylls, carotenoids, and hopanoids, are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate. Isoprenoid biosynthesis in extracts of the cyanobacterium Synechocystis strain PCC 6803 grown under photosynthetic conditions, stimulated by pentose phosphate cycle substrates, does not appear to require methylerythritol phosphate pathway intermediates. The sll1556 gene, distantly related to type 2 IPP isomerase genes, was disrupted by insertion of a Kan r cassette. The mutant was fully viable under photosynthetic conditions although impaired in the utilization of pentose phosphate cycle substrates. Compared to the parental strain the ⌬sll1556 mutant (i) is deficient in isoprenoid biosynthesis in vitro with substrates including glyceraldehyde-3-phosphate, fructose-6-phosphate, and glucose-6-phosphate; (ii) has smaller cells (diameter ca. 13% less); (iii) has fewer thylakoids (ca. 30% less); and (iv) has a more extensive fibrous outer wall layer. Isoprenoid biosynthesis is restored with pentose phosphate cycle substrates plus the recombinant Sll1556 protein in the ⌬sll1556 supernatant fraction. IPP isomerase activity could not be demonstrated for the purified Sll1556 protein under our in vitro conditions. The reduction of thylakoid area and the effect on outer wall layer components are consistent with an impairment of isoprenoid biosynthesis in the mutant, possibly via hopanoid biosynthesis. Our findings are consistent with an alternate metabolic shunt for biosynthesis of isoprenoids.Isoprenoids are required for many cell processes including photosynthesis, membrane stability, electron transport, and the cellular production of carotenoids, rubber, and fragrances. More than 30,000 isoprenoid compounds have been described. Synthesis of the essential 5-carbon building blocks of isoprenoids, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), can be attributed to one of two pathways. The mevalonic acid pathway, the sole pathway in animals and in many bacteria, also occurs in the cytoplasm of plant cells, where it is responsible for synthesis of sterols and ubiquinones (18,20). The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway occurs in Escherichia coli and many other bacteria as well as in cyanobacteria. It is also present in chloroplasts, where it provides substrates for the synthesis of carotenoids, chlorophylls, and quinones. This pathway is believed important for cyanobacterial photosynthetic pigment biosynthesis including that of carotenoids and the phytolation of chlorophyll and in hopanoid synthesis of bacterial and cyanobacterial cell walls and membranes (8,20,24). Most of the MEP pathway genes have been functionally verified in the gram-negative heterotrophic bacterium E. coli (reviewed in references 4, 14, 18, and 20), and this bacterium has thus become the standard for defining the MEP pathway in other organisms. The MEP pathway is typically represented as a linear sequence of reactions commenci...