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 CO 2 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-CO 2-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 CO 2 level and the double mutant ⌬glcD1/⌬glcD2 was unable to grow under low CO2. The HCR phenotype of both the double and the triple mutant could not be attributed to a significantly reduced affinity to CO2, such as in other cyanobacterial HCR mutants defective in the CO2-concentrating mechanism (CCM). These unexpected findings of an HCR phenotype in the presence of an active CCM indicate that 2PG metabolism is essential for the viability of all organisms that perform oxygenic photosynthesis, including cyanobacteria and C3 plants, at ambient CO 2 conditions. These data and phylogenetic analyses suggest cyanobacteria as the evolutionary origin not only of oxygenic photosynthesis but also of an ancient photorespiratory 2PG metabolism. I t is well established that the photorespiratory C2 pathway, whereby 2-phosphoglycolate (2PG) is metabolized (1), is essential for photosynthesis in the majority of plants (2). In contrast, the functioning of the C2 pathway and its importance are still under discussion for cyanobacteria. These organisms were the first to have evolved oxygenic photosynthesis, and endosymbiotic engulfment of an ancient cyanobacterium led to the evolution of plant chloroplasts (3). In cyanobacteria, as in C3 plants, the primary carbon fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Ribulose 1,5-bisphosphate reacts with either CO 2 , leading to the formation of 2 molecules of 3-phosphoglycerate (3PGA), or O 2 , generating 3PGA and 2PG. The latter compound is toxic to plant metabolism because it inhibits distinct steps in the carbon-fixing Calvin-Benson cycle (4, 5). Therefore, plants employ the socalled photorespiratory glycolate pathway (or C2 cycle), which degrades 2PG and converts 2 molecules of 2PG into 1 molecule each of 3PGA, CO 2 , and NH 4 ϩ (1, 6, 7). In a typical C3 plant, the ammonium is refixed at the expense of ATP, and 25% of the carbon entering the path is released as CO 2 . Generally, the photorespiratory cycle is indispensable for C3 plants, because mutations in single steps of the C2 cycle resulted in high-CO 2 -requiring (HCR) phenotypes (2,(8)(9)(10).In contrast to plants, ear...

Understanding the behavior of ionic liquids on the molecular level is essential for explaining solubilizing or reaction processes, including catalytic reactions in ionic liquids or with ionic liquids as co-solvent. Using mass spectrometry techniques it is possible to characterize their aggregate formation behavior, which depends on the used solvent. With increasing polarity of the solvent and decreasing ionic liquid concentration, the size of the formed aggregates decreases. From conductivity measurement curves "critical aggregate concentrations" were calculated, which confirm the results of mass spectrometry measurements. Addition of ionic liquids increases the solubility of acetophenone in water. This effect can be explained by the aggregate formation ability of ionic liquids. The findings can be used to explain the outstanding solubility and solvation properties of ionic liquids.

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO 2 -requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacteriallike glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO 2 . Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO 2 . These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO 2 , glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.

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Compatible solutes are small molecules that are involved in acclimation to various abiotic stresses, especially high salinity. Among the red algae, the main photosynthetic products floridoside and isofloridoside (galactosylglycerols) are known also to contribute to the osmotic acclimation of cells. However, the genes encoding (iso)floridoside biosynthetic enzymes are still unknown. To identify candidate genes, we examined the genome of the floridoside- and isofloridoside-accumulating extremophilic red alga Galdieria sulphuraria belonging to the Cyanidiales. We hypothesized that two candidate genes, Gasu_10960 and Gasu_26940, code for enzymes involved in floridoside and isofloridoside biosynthesis. These proteins comprise a sugar phosphate synthase and a sugar phosphate phosphatase domain. To verify their biochemical activity, both genes were in vitro translated into the entire proteins. The protein translation mixture containing Gasu_10960 synthesized small amounts of isofloridoside, whereas the Gasu_26940 translation mix also produced small amounts of floridoside. Moreover, the expression of Gasu_10960 in a salt-sensitive mutant of the cyanobacterium Synechocystis sp. PCC 6803 resulted in increased salt tolerance as a consequence of the presence of isofloridoside in the complemented cells. Thus, our experiments suggest that the Gasu_26940 and Gasu_10960 genes of G. sulphuraria encode the enzymatically active floridoside and isofloridoside phosphate synthase/phosphatase fusion proteins, respectively, crucial for salt acclimation.

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The polysaccharide laminarin (β-1,3-glucan) is used as a long-term carbon storage compound in brown algae. This chemical storage form of carbon enables perennial brown algae in seasonally fluctuating ecosystems to uncouple growth from photosynthesis, i.e., most of these plants grow as seasonal anticipators in winter based on remobilization of laminarin, while in summer, growth typically ceased to fill up the storage pool. Because of this high ecological relevance, a reliable and precise method for determination and quantification of laminarin is needed. Therefore, a simple, efficient, cold water extraction method coupled to a new quantitative liquid chromatography-mass spectrometrical method (LC-MS) was developed. Laminarin was determined in 9 out of 12 brown algal species, and its expected typical molar mass distribution of 2000-7000 Da was confirmed. Furthermore, laminarin consisted of a complex mixture of different chemical forms, since 15 chemical laminarin species with distinct molecular weights were measured in 9 species of brown algae. Differences in chain length and number of laminarin species seem to be species specific and hence may indicate some chemotaxonomic value. Laminarin concentrations in the algal tissues ranged from 0.03 to 0.86 % dry weight (DW). The direct chemical characterization and quantification of laminarin by LC-MS represents a powerful method to verify the biochemical and ecological importance of laminarin for brown algae.

Pervaporation proved to be one of the best methods to remove solvents out of a solvent producing Clostridium acetobutylicum culture. By using an ionic liquid (IL)-polydimethylsiloxane (PDMS) ultrafiltration membrane (pore size 60 nm), we could guarantee high stability and selectivity during all measurements carried out at 37°C. Overall solvent productivity of fermentation connected with continuous product removal by pervaporation was 2.34 g l −1 h −1 . The supported ionic liquid membrane (SILM) was impregnated with 15 wt% of a novel ionic liquid (tetrapropylammonium tetracyano-borate) and 85 wt% of polydimethylsiloxane. Pervaporation, accomplished with the optimized SILM, led to stable and efficient removal of the solvents butan-1-ol and acetone out of a C. acetobutylicum culture. By pervaporation through SILM, we removed more butan-1-ol than C. acetobutylicum was able to produce. Therefore, we added an extra dose of butan-1-ol to run fermentation on limiting values where the bacteria would still be able to survive its lethal concentration (15.82 g/l). After pervaporation was switched off, the bacteria died from high concentration of butan-1-ol, which they produced.