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.

     

Glutamatdehydrogenase im photosynthetischen Bakterium Rhodospirillum rubrum

Zusammenfassung Glutamatdehydrogenase wurde aus Rhodospirillum rubrum durch Fällung mit Ammoniumsulfat und Chromatographie an DEAE-Cellulose 35 fach angereichert. Das Enzym ist spezifisch auf NAD als Wasserstoffdonator/acceptor und -Ketoglutarat bzw. Glutamat. Hg-Ionen blockieren die Reaktion in beiden Richtungen; Nitrit- und Nitrationen hemmen in höheren Konzentrationen. Die Abbaurate wird durch die Anwesenheit von ATP verringert. Die Stickstoffquelle des Nährmediums wirkt sich nur wenig auf die Ausbildung des Enzyms in den Zellen aus, dagegen wird durch Produkthemmung im natürlichen Milieu bei Wachstum auf Malat und NH₄ ⁺ der Glutamatabbau praktisch unterdrückt.

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Glutamate dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum

Summary Glutamate dehydrogenase from Rhodospirillum rubrum was purified 35 fold by ammonium sulfate precipitation and chromatography on DEAE-cellulose. The enzyme is specific for NAD as hydrogen donor/acceptor and -ketoglutarate and glutamate for the synthesis, respectively the degradation of the amino acid. Hg²⁺ ions completely inhibit both synthesis and degradation; a weaker inhibition can be shown by addition of various inorganic nitrogen compounds. The rate of the glutamate degradation is reduced by ATP. The nitrogen source of the culture medium is without effect on the formation of the glutamate dehydrogenase, however, under growth conditions in a malate-NH₄ ⁺-solution the glutamate degradation is almost completely suppressed by product inhibition.

     

Carbon monoxide dehydrogenase from Rhodospirillum rubrum

The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 microM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.     

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.     

Ein neues Bacteriochlorophyll aus Rhodospirillum rubrum

Zusammenfassung Rhodospirillum rubrum und Rhodopseudomonas palustris enthalten ein Bacteriochlorophyll, das mit Farnesol statt mit Phytol veretert ist. Das neue Bacteriochlorophyll (Bchl a_F) läßt sich vom bekannten Bacteriochlorophyll a (Bchl a_P) durch ¹H-NMR-Spektroskopie unterscheiden sowie durch Dünnschichtchromatographie der entsprechenden Phäophytine an mit Silbernitrat imprägniertem Kieselgel. Die Chromophore von Bchl a_F und Bchl a_P sind auch bezüglich ihrer Stereochemie identisch.

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A new bacteriochlorophyll from Rhodospirillum rubrum

Summary Rhodospirillum rubrum and Rhodopseudomonas palustris contain a bacteriochlorophyll which is a farnesyl rather than a phytyl ester. The new bacteriochlorophyll (Bchl a_F) can be distinguished from the well known bacteriochlorophyll a (Bchl a_P) by ¹H-n.m.r. spectroscopy and by t.l.c. of the corresponding pheophytins on silver nitrate impregnated silica gel. The chromophores of Bchl a_P and Bchl a_F are identical in structure and stereochemistry.

     

Studies in the phototaxis of Rhodospirillum rubrum

Summary The threshold strength-duration relationships were determined for the phototactic excitation of Rhodospirillum rubrum by various pulses and pairs of pulses of change in light intensity. The recovery of excitability after a response was followed, and examples of rhythmic behavior were recorded.Exprimental results were found to be in fair agreement with data for other irritable systems and with the predictions of the theories of Rashevsky and Hill.The hypothesis was considered that all excitable systems might share a common mechanism for irritability, and the phototactic mechanisms of various unicellular organisms were discussed in this connection.     

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.     

Whole-genome shotgun optical mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a "molecular cytogenetics" approach to solving problems in genomic analysis.     

Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

Rhodospirillum rubrum is a phototrophic purple nonsulfur bacterium known for its unique and well-studied nitrogen fixation and carbon monoxide oxidation systems and as a source of hydrogen and biodegradable plastic production. To better understand this organism and to facilitate assembly of its sequence, three whole-genome restriction endonuclease maps (XbaI, NheI, and HindIII) of R. rubrum strain ATCC 11170 were created by optical mapping. Optical mapping is a system for creating whole-genome ordered restriction endonuclease maps from randomly sheared genomic DNA molecules extracted from cells. During the sequence finishing process, all three optical maps confirmed a putative error in sequence assembly, while the HindIII map acted as a scaffold for high-resolution alignment with sequence contigs spanning the whole genome. In addition to highlighting optical mapping's role in the assembly and confirmation of genome sequence, this work underscores the unique niche in resolution occupied by the optical mapping system. With a resolution ranging from 6.5 kb (previously published) to 45 kb (reported here), optical mapping advances a “molecular cytogenetics” approach to solving problems in genomic analysis.     

Carbon monoxide-dependent growth of Rhodospirillum rubrum.

Under dark, anaerobic conditions in the presence of sufficient nickel, Rhodospirillum rubrum grows with a doubling time of under 5 h by coupling the oxidation of CO to the reduction of H+ to H2. CO-dependent growth of R. rubrum UR294, bearing a kanamycin resistance cassette in cooC, depends on a medium nickel level ninefold higher than that required for optimal growth of coo+ strains.