Inactivation ofsll1556inSynechocystisStrain PCC 6803 Impairs Isoprenoid Biosynthesis from Pentose Phosphate Cycle Substrates In Vitro

Abstract

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...

Isolation and identification of a new antibacterial entity from the Antarctic cyanobacterium Nostoc CCC 537

“…The inherent capacity to produce specific bioactive compounds/secondary metabolites seems to play a vital role in colonisation of Nostoc in tropical and Antarctic regions (Dodds et al 1995). The observed difference in extent of secondary metabolite production in cyanobacteria could be attributed to the environmentally induced utilisation of metabolite intermediates (Poliquin et al 2004). The hyperaccumulation of unsaturated fatty acids by the Antarctic strains (Wada et al 1990(Wada et al , 1994Los et al 1993;Vigh et al 1993) has been linked with high synthesis of secondary metabolites, probably through breakdown of fatty acids during the stationary phase of growth.…”

The Methylerythritol Phosphate Pathway Is Functionally Active in All Intraerythrocytic Stages of Plasmodium falciparum

“…Several hypotheses could be suggested to explain the low level of inhibition of the biosynthesis of MEP, CDP-ME, CDP-MEP, ME-2,4-cPP, dolichols, and the isoprenic chain of ubiquinones detected in trophozoite and schizont stages obtained from asynchronous cultures treated with fosmidomycin: (a) an alternative pathway for the biosynthesis of IPP and dimethylallyl diphosphate could be active in which xylulose 5Ј-phosphate is considered as a precursor (72,73); and (b) if P. falciparum performs active de novo biosynthesis of thiamin from DOXP, the levels of thiamin diphosphate could be increased in fosmidomycin-treated parasites, as we demonstrated for pyridoxine 5Ј-phosphate. Because 1-deoxy-D-xylulose-5-phosphate synthase is thiamin diphosphate-dependent, its enzymatic activity could be modulated and, as a consequence, the downstream enzymes from the MEP pathway, too.…”

Inactivation of Genes Encoding Plastoglobulin-Like Proteins in Synechocystis sp. PCC 6803 Leads to a Light-Sensitive Phenotype

“…Electron microscopy. Samples used for transmission electron microscopy were fixed, dehydrated, and embedded in Epon resin as described by Poliquin et al (29). Some of the glutaraldehyde-fixed samples (which were not secondarily fixed with OsO 4 ) were dehydrated and embedded in LR White resin rather than Epon, and polymerization was catalyzed by UV light at Ϫ20°C.…”