Photosynthetic metabolism of propionate in Rhodospirillum rubrum

Summary As in the case of oxidative metabolism, the photosynthetic metabolism of propionate in Rhodospirillum rubrum begins with a carboxylation yielding succinate. This conclusion is based on experiments in which radioactive propionate (1-C¹⁴ and 2-C¹⁴) is administered in the presence of carrier lactate, pyruvate, succinate, and acrylate, and on studies of the inhibitory action of malonate.     

Competition between light and dark metabolism in Rhodospirillum rubrum

Summary In the presence of both light and air, the metabolism of Rhodospirillum rubrum can be partly respiratory and partly photosynthetic. The relative rates of these modes of metabolism have been measured at a variety of light intensities and oxygen tensions.     

The Structure of Rhodospirillum rubrum

Cells from serial cultures of R. rubrum, grown anaerobically in the light, were harvested at intervals from ½ to 15 days and sectioned for electron microscopy by conventional methods. Cells of this species possess a multilayered outer envelope, and the external cell surface is differentiated into ridges extending parallel or obliquely to the long axis of the cell. Cells from very young cultures resemble non-photosynthetic bacteria and contain only a granular cytoplasm, scattered high-density particles, and low-density areas corresponding to the chromatin areas observed by light microscopy. They contain neither the chromatophores nor the lamellar systems assumed by previous investigators to be characteristic of this species when grown anaerobically in the light. Chromatophores appear in cells from cultures older than about 12 hours, while systems of paired lamellae appear along with the chromatophores in cells from cultures older than about 8 days. Divergent opinions concerning the occurrence of chromatophores or lamellae in this species can be resolved on the basis of the age of cultures used in previous studies. Other changes occurring in cells from cultures of increasing age include the appearance of granular and reticulate cytoplasmic bodies and vacuoles, extension of the chromatin areas, and the appearance of a single membrane enclosing several chromatophores.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Fermentative metabolism of pyruvate by Rhodospirillum rubrum after anaerobic growth in darkness.

Rhodospirillum rubrum grew anaerobically in darkness and fermented sodium pyruvate by a pyruvate formate-lyase reaction. During 30 min of anaerobic dark or light incubation with sodium pyrivate, crude extracts from fermentatively grown cells produced about 6 micronmol of acetylphosphate and formate per mg of protein in reactions performed at pH 8.3. Cell extracts also catalyzed the exchange of sodium [14C]formate into sodium pyruvate at an apparent pH optimum of 7.3 to 7.5, but only about 2.5 micronmol of acetylphosphate was produced at this lower pH value. R. rubrum may also form pyruvate:ferredoxin oxidoreductase activity, as evidenced by low bicarbonate exchange activity. However, its participation in pyruvate metabolism in anaerobic dark-grown cells was not understood. During anaerobic, dark growth with pyruvate, formate was an intermediate in H2 and CO2 gas evolution. In contrast with H2 production by a light-dependent H2-nitrogenase system in photosynthetically grown cells, H2 formation in fermenting R. rubrum occurred through a carbon monoxide-sensitive formic hydrogenlyase reaction not influenced by light.     

Ferredoxin-dependent carbon assimilation in Rhodospirillum rubrum

Summary Evidence has been presented that a soluble fraction from R. rubrum cells contains two new primary carboxylation reactions which depend on the reducing power of ferredoxin: (a) pyruvate synthase which brings about a synthesis of pyruvate from acetyl-CoA and CO₂ and (b) -ketoglutarate synthase which brings about a synthesis of -ketoglutarate from succinyl-CoA and CO₂. The soluble fraction of R. rubrum cells contains also a series of other enzymes which, together with the ferredoxin-dependent enzymes, constitutes a reductive carboxylic acid cycle—a new cyclic pathway for CO₂ assimilation that was first found in the photosynthetic bacterium, Chlorobium thiosulfatophilum.Dedicated to C. B. van Niel on the occasion of his 70th birthday.Aided by grants from the National Institute of General Medical Sciences, Office of Naval Research and the National Science Foundation.     

Microaerophilic Cooperation of Reductive and Oxidative Pathways Allows Maximal Photosynthetic Membrane Biosynthesis in Rhodospirillum rubrum

The purple nonsulfur bacterium Rhodospirillum rubrum has been employed to study physiological adaptation to limiting oxygen tensions (microaerophilic conditions). R. rubrum produces maximal levels of photosynthetic membranes when grown with both succinate and fructose as carbon sources under microaerophilic conditions in comparison to the level (only about 20% of the maximum) seen in the absence of fructose. Employing a unique partial O₂ pressure (pO₂) control strategy to reliably adjust the oxygen tension to values below 0.5%, we have used bioreactor cultures to investigate the metabolic rationale for this effect. A metabolic profile of the central carbon metabolism of these cultures was obtained by determination of key enzyme activities under microaerophilic as well as aerobic and anaerobic phototrophic conditions. Under aerobic conditions succinate and fructose were consumed simultaneously, whereas oxygen-limiting conditions provoked the preferential breakdown of fructose. Fructose was utilized via the Embden-Meyerhof-Parnas pathway. High levels of pyrophosphate-dependent phosphofructokinase activity were found to be specific for oxygen-limited cultures. No glucose-6-phosphate dehydrogenase activity was detected under any conditions. We demonstrate that NADPH is supplied mainly by the pyridine-nucleotide transhydrogenase under oxygen-limiting conditions. The tricarboxylic acid cycle enzymes are present at significant levels during microaerophilic growth, albeit at lower levels than those seen under fully aerobic growth conditions. Levels of the reductive tricarboxylic acid cycle marker enzyme fumarate reductase were also high under microaerophilic conditions. We propose a model by which the primary “switching” of oxidative and reductive metabolism is performed at the level of the tricarboxylic acid cycle and suggest how this might affect redox signaling and gene expression in R. rubrum.     

Glycolate excretion by Rhodospirillum rubrum

Glycolate can be measured in the supernatant fraction after incubation of butyrate-grown cells of Rhodospirillum rubrum either colorimetrically by the Calkins method or enzymatically using glycolate oxidase. Under optimal conditions, half-maximal excretion occurs at 11% O₂ and the maximal rate is 6.9 nmol of glycolate min^-1 mg protein^-1 at 30°C. The pH and temperature optima are 7.6 and 30°C and light intensity is saturating in the range of 2–10×10⁴ lux. Carbon dioxide inhibits glycolate excretion and exogenous butyrate stimulates. Glycolate excretion is maximal by butyrate-light grown cells harvested in the early stationary phase and under all conditions is proportional to the cellular content of ribulose 1,5-bisphosphate carboxylase/oxygenase.Non-Standard Abbreviations Bicine (N,N-bis[2-hydroxyethyl]glycine) - RuBP d-ribulose-1,5-bisphosphate - HPMS 2-pyridylhydroxymethanesulfonate     

Glutathione reductase from Rhodospirillum rubrum

Summary Glutathione reductase (NADPH¹: glutathione oxidoreductase (EC 1.6.4.2) was purified 70 fold from Rhodospirillum rubrum by ammonium sulfate fractionation, gelfiltration with Sephadex and chromatography on DEAE-cellulose. The optimum pH of the reaction is 7.5–8.2 K _m values of 8.4×10^–6 M for NADPH and 5.8×10^–5 M for GSSG were determined. The kinetic data indicate a bisubstrate reaction mechanism. The prosthetic group is FAD (K _m 1.1×10^–6M). The flavin can be completely dissociated from the enzyme, and 70% of the original activity can subsequently be restored by FAD. The molecular weight was determined with a calibrated column Sephadex G-200 and found to be approximately 63,000. The enzyme is inhibited reversibly by several anions. With iodide the inhibition is competitive with respect to GSSG. Sulfhydryl reagents (N-ethylmaleinimide, p-chlormercuribenzoate) strongly inhibit the enzyme when it is present in the reduced state. The enzyme is reduced by low concentrations of NADPH and by higher concentrations of NADH. GSSG protects the enzyme against this inhibition. The enzyme is reversibly inhibited by incubation with NADPH or NADH.

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Zusammenfassung Glutathionreduktase wurde aus Rhodospirillum rubrum mit Ammoniumsulfatfraktionierung, Gelfiltration mit Sephadex und Chromatographie an DEAE-Cellulose 70 fach angereichert. Das pH Optimum der Reaktion liegt bei 7,5–8,2. K _m -Werte: 8,4·10^–6 M für NADPH und 5,8·10^–5 M für GSSG. Aus den kinetischen Daten ergibt sich für das Enzym ein Bisubstratreaktionsmechanismus. Die prosthetische Gruppe ist FAD (K _m 1,1·10^–6 M). Das Flavin kann vollständig vom Enzymprotein abdissoziiert werden, durch erneute Zugabe von FAD können etwa 70% der ursprünglichen Aktivität zurückerhalten werden. Das Molekulargewicht, bestimmt durch Gelfiltration mit einer kalibrierten Säule Sephadex G-200, ist ca. 63000. Das Enzym wird durch verschiedene Anionen reversibel gehemmt. Bei J^– ist die Hemmung kompetitiv mit GSSG. Sulfhydrylreagentien (N-Äthylmaleinimid und p-Chlomercuribenzoat) sind potente Inhibitoren, wenn das Enzym im reduzierten Zustand vorliegt. Das Enzym kann bereits durch niedrige Konzentrationen an NADPH sowie durch höhere Konzentrationen an NADH reduziert werden. GSSG schützt das Enzymprotein gegen die Hemmung durch Sulfhydryl-reagentien. Das Enzym wird durch Inkubation mit NADPH und NADH reversibel gehemmt.