Wolfgang Ruth

The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants

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 ΔglcD1/ΔglcD2 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 CO2 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.

The Plant-Like C2 Glycolate Cycle and the Bacterial-Like Glycerate Pathway Cooperate in Phosphoglycolate Metabolism in Cyanobacteria

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-CO2-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 bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO2. 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 CO2. These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO2, 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.

Valinomycin mediated transfer of potassium across the water/nitrobenzene interface: A study by voltammetry at the interface between two immiscible electrolyte solutions*

The valinomycin-mediated transfer of potassium ions across the water/nitrobenzene interface was studied by potential-sweep voltammetry at this interface (ITIES). With a sweep rate 16.3 mV s−1 and at low concentrations of valinomycin reversible peak voltammograms were obtained. The charge transfer process is controlled by diffusion of valinomycin to ITIES and of the complex from ITIES into the bulk of the organic phase. The formation and the dissociation of the complex are rapid surface reactions. From the peak potential the stability constant of the valinomycin-potassium complex log KKV+=5.5 was determined.

Diversity of ion carrier functions of monensin: a study using voltammetry at the interface of two immiscible electrolyte solutions

Abstract Sodium and hydrogen ion transfer across the water/nitrobenzene interface in the presence of monensin (HX) was studied using voltammetry at the interface of two immiscible electrolyte solutions (ITIES). Among the monensin species present in the system, HX functions as a sodium carrier and NaX as a proton carrier, the species transferred in both cases being NaHX+. This species is formed in nitrobenzene either by the reaction Na++HX NaHX+ (equilibrium constant log K1=5.63) or by the reaction H++NaX NaHX+ (equilibrium constant log K2=9.88). The principal electroneutral process is the exchange reaction Na+(w)+HX(o) NaX(o)+H+(w) (w=water, o=nitrobenzene) with the equilibrium constant log Kexch=−4.54.

Assessment of acetone as an alternative to acetonitrile in peptide analysis by liquid chromatography/mass spectrometry

Acetonitrile as a solvent used in liquid chromatography/mass spectrometry (LC/MS) of peptides and proteins is a relatively toxic solvent (LD50 oral; rat; 2,460 mg/kg) compared to alternatives like methanol (LD50 oral; rat; 5,628 mg/kg) and acetone (LD50 oral; rat; 5,800 mg/kg). Strategies to minimize its consumption in LC are either to reduce the inner diameter of the column or replace acetonitrile with a suitable alternative. Methanol is often recommended to replace acetonitrile in peptide analysis. In this study however, the main focus lies on another alternative solvent for LC/MS of peptides; acetone. A number of model proteins were tryptically digested and the peptide solutions were analyzed on a linear trap quadrupole (LTQ) mass spectrometer. The performances of acetonitrile, methanol and acetone were compared according to the quality of the chromatograms obtained and identification of the peptides using the BioWorks software developed by Thermo Scientific. In accordance to the elutropic series, acetone was found to significantly reduce the retention times of peptides separated by C18 column material with regard to acetonitrile while methanol led to increased retention times. Acetone was the superior solvent to methanol for most of the tested model proteins reaching similar sequence coverage and numbers of identified peptides as acetonitrile. We therefore propose acetone as an alternative to acetonitrile in LC/MS of peptides.

Determination of Monensin by Voltammetry at the Interface between Two Immiscible Electrolyte Solutions

Abstract The coccidiostat monensin can be determined by voltammetry at the interface between two immiscible electrolyte solutions (ITIES) in the concentration range between 0.05 mM to 3 mM. The peaks obtained with cyclic voltammetry at the water/nitrobenzene interface correspond to the transfer of sodium ions from the aqueous to the nonaqueous phase facilitated by complexation with monensin functioning as an ionophore and are proportional to its concentrations. The method was applied to the extracts from Streptomyces cultures.

Effects of Phytoestrogen Extracts Isolated from Rye, Green and Yellow Pea Seeds on Hormone Production and Proliferation of Trophoblast Tumor Cells Jeg3

Background: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. Objectives: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. Methods: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. Results: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. Conclusion: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Transfer of alkali-metal and hydrogen ions across liquid/liquid interfaces mediated by monensin. A voltammetric study at the interface of two immiscible electrolyte solutions

Under certain conditions the interface between an aqueous phase and an organic phase (ITIES, interface of two immiscible electrolyte solutions) has certain properties analogous to a metal/electrolyte-solution interface. By polarization of the ITIES using potential-sweep voltammetry reproducible voltammograms corresponding to ion transfer across the ITIES are obtained. In the presence of cation-complexing ionophores in the organic phase cation transfer is facilitated. The acidic form HX of carboxylic ionophores, monensin A and B, acts in the nitrobenzene/water system as a sodium carrier while the complex of its anion with sodium or lithium cation (M+) is a proton carrier. The equilibrium constants of the reactions M++ HX ⇌ MHX+ and MX + H+⇌ MHX+ have been determined from the voltammograms. The stabilities of the complexes correspond to the series Li+ > Na+≫ K+. The substitution of methyl (monensin B) for ethyl (monensin A) on ring C increases the stability of the sodium complex by ca. 1 kJ mol–1 and has no effect on the acidity of the carboxy group.

Season exerts differential effects of ocean acidification and warming on growth and carbon metabolism of the seaweed Fucus vesiculosus in the western Baltic Sea

Warming and acidification of the oceans as a consequence of increasing CO2-concentrations occur at large scales. Numerous studies have shown the impact of single stressors on individual species. However, studies on the combined effect of multiple stressors on a multi-species assemblage, which is ecologically much more realistic and relevant, are still scarce. Therefore, we orthogonally crossed the two factors warming and acidification in mesocosm experiments and studied their single and combined impact on the brown alga Fucus vesiculosus associated with its natural community (epiphytes and mesograzers) in the Baltic Sea in all seasons (from April 2013 to April 2014). We superimposed our treatment factors onto the natural fluctuations of all environmental variables present in the Benthocosms in so-called delta-treatments. Thereby we compared the physiological responses of F. vesiculosus (growth and metabolites) to the single and combined effects of natural Kiel Fjord temperatures and pCO2 conditions with a 5 °C temperature increase and/or pCO2 increase treatment (1100 ppm in the headspace above the mesocosms). Responses were also related to the factor photoperiod which changes over the course of the year. Our results demonstrate complex seasonal pattern. Elevated pCO2 positively affected growth of F. vesiculosus alone and/or interactively with warming. The response direction (additive, synergistic or antagonistic), however, depended on season and daylength. The effects were most obvious when plants were actively growing during spring and early summer. Our study revealed for the first time that it is crucial to always consider the impact of variable environmental conditions throughout all seasons. In summary, our study indicates that in future F. vesiculosus will be more affected by detrimental summer heat-waves than by ocean acidification although the latter consequently enhances growth throughout the year. The mainly negative influence of rising temperatures on the physiology of this keystone macroalga may alter and/or hamper its ecological functions in the shallow coastal ecosystem of the Baltic Sea.

Determination of aniline traces in nitrobenzene by the facilitated proton transfer across the water/nitrobenzene interface

Abstract The proposed analytical method is based on the voltammetrical investigation of the interface between two immiscible electrolyte solutions. Transfer of different ions across the interface between water and nitrobenzene can be observed by using cyclic voltammetry. Facilitated transfer of protons from aqueous into nitrobenzene phase can be observed if there is present a suitable organic base like, e.g., aniline in the nonaqueous media. From the peak current the amount of aniline in nitrobenzene can be established. The lowest measurable concentration was 5 × 10−5 M 4.56 mg of aniline per liter of nitrobenzene.