Nucleotide sequence of the Rhodospirillum rubrum atp operon.

The nucleotide sequence was determined of a 8775-base-pair region of DNA cloned from the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. It contains a cluster of five genes encoding F1-ATPase subunits. The genes are arranged in the same order as F1 genes in the Escherichia coli unc operon. However, as in the related organism Rhodopseudomonas blastica, neither genes for components of F0, the membrane sector of ATP synthase, nor a homologue of the E. coli uncI gene are associated with this locus, as they are in E. coli.     

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.     

The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex

Summary A cytochrome bc ₁-complex of Rs. rubrum was isolated and the three subunits were purified to homogeneity. The N-terminal amino acid sequence of the purified subunits was determined by automatic Edman degradation. The pet genes of Rhodospirillum rubrum coding for the three subunits of the cytochrome bc ₁-complex were isolated from a genomic library of Rs. rubrum using oligonucleotides specific for conserved regions of the subunits from other organisms and a heterologous probe derived from the genes for the complex of Rb. capsulatus. The complete nucleotide sequence of a 5500 by SalI/SphI fragment is described which includes the pet genes and three additional unidentified open reading frames. The N-terminal amino acid sequence of the isolated subunits was used for the identification of the three genes. The genes encoding the subunits are organized as follows: Rieske protein, cytochrome b, cytochrome c ₁. Comparison of the N-terminal protein sequences with the protein sequences deduced from the nucleotide sequence showed that only cytochrome c ₁ is processed during transport and assembly of the three subunits of the complex. Only the N-terminal methionine of the Rieske protein is cleaved off. The similarity of the deduced amino acid sequence of the three subunits to the corresponding subunits of other organisms is described and implications for structural features of the subunits are discussed.Abbreviations BSA bovine serum albumin - SDS sodium dodecylsulphate - Rs Rhodospirillum - Rb Rhodobacter - Pc Paracoccus - Rps Rhodopseudomonas The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X55387     

Characterization of altered regulation variants of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum

In Rhodospirillum rubrum, nitrogenase activity is subject to posttranslational regulation through the adenosine diphosphate (ADP)-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT) and dinitrogenase reductase-activating glycohydrolase (DRAG). To study the posttranslational regulation of DRAG, its gene was mutagenized and colonies screened for altered DRAG regulation. Three different mutants were found and the DRAG variants displayed different biochemical properties including an altered affinity for divalent metal ions. Taken together, the results suggest that the site involved in regulation is physically near the metal binding site of DRAG.     

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.     

Untersuchungen zur Photophosphorylierung bei Rhodospirillum molischianum und Rhodospirillum rubrum

Zusammenfassung Die Thylakoide aus Rhodospirillum rubrum und Rhodospirillum molischianum werden nach Homogenisation der Zellen mit Ultraschall durch fraktionierte Zentrifugation isoliert. An diese Membranstrukturen ist das System der Photophosphorylierung gebunden. Die Aktivität dieses Systems in Abhängigkeit vom Redoxpotential des Mediums wird untersucht. Die stärkste Bindung anorganischen Phosphates wird unter Edelgasatmosphäre bei Zusatz von Spuren eines Elektronendonators (0,07 mol Succinat je Ansatz) beobachtet. Die cyclische Photophosphorylierung wird einerseits durch Sauerstoff und oxydierende Verbindungen wie K₃Fe(CN)₆ anderseits durch Überreduktion mittels reduzierter Redoxverbindungen wie 2,6-Dichlorphenolindophenol oder Phenazinmethosulfat (beide reduziert durch Ascorbat) unter Wasserstoffatmosphäre gehemmt. Die Sauerstoffhemmung kann durch reduziertes Phenazinmethosulfat zu 50% aufgehoben werden. Antimycin A blockiert die lichtabhängige Phosphorylierung; 2,4-Dinitrophenol dagegen hemmt kaum. Die zellfreien Systeme beider Arten zeigen die gleiche Abhängigkeit vom Redoxpotential obwohl R. rubrum wesentlich sauerstofftoleranter ist als R. molischianum und auch durch oxydative Phosphorylierung im Dunkeln ATP bilden kann. Die Befunde sprechen für eine Unabhängigkeit der cyclischen Photophosphorylierung von der Atmungskette und für eine starke Übereinstimmung im Aufbau der Elektronentransportsysteme für die cyclische Photophosphorylierung bei R. rubrum und R. molischianum.

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Summary The isolated thylakoide-structures (chromatophores) of Rhodospirillum molischianum and Rhodospirillum rubrum are investigated with regard to activity of cyclic, light induced phosphorylation. A high activity of the photochemical apparatus needs an optimal external oxidation-reduction potential. Over-oxidation by oxygen or K₃[Fe[CN)₆] inhibit just as much as over-reduction by hydrogen and N-methyl phenazonium methosulfate or 2,6-dichlorphenol-indophenole both reduced with ascorbate. The highest activity is observed in hydrogen atmosphere without an electron-donator system or in helium with traces (0,07 mol) of succinate. The inhibitoryeffect of oxygen can partly be compensated by reduced PMS. The photochemical apparatus of R. molischianum and R. rubrum react nearly in the same way on changes of the external oxidation-reduction potential and both systems are strongly inhibited by antimycin A but not by low concentrations of 2,4-dinitrophenole. R. molischianum is a strict anaerobic organism and can grow only in the light and under low oxygen partialpressure. In comparison with it R. rubrum is more oxygen-tolerant. The strain can grow under conditions of aerobic dark metabolism. The present results together with those of other investigations provide strong evidence for the conclusion that the systems of cyclic photophosphorylation in both organisms have the same composition and are relatively independent from the respiratory chain.

     

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.

     

Characterization of the interaction of dinitrogenase reductase-activating glycohydrolase from Rhodospirillum rubrum with bacterial membranes

The interaction of dinitrogenase reductase-activating glycohydrolase (DRAG) with bacterial membranes and the solubilization of DRAG in response to nucleotides were characterized. Purified DRAG from Rhodospirillum rubrum reversibly bound bacterial pellet fractions from Rsp. rubrum and other nitrogen-fixing bacteria. DRAG saturated the membrane fraction of Rsp. rubrum at a concentration of 0.2 mol DRAG/mol bacteriochlorophyll, suggesting that the DRAG-binding species is prevalent in the membrane. DRAG bound poorly to phospholipid vesicles, suggesting a protein requirement for DRAG interaction with the membrane. Guanosine and uridine tri- and di-nucleotides specifically dissociated DRAG from the pellet fractions of Rsp. rubrum and Azotobacter vinelandii, while adenosine nucleotides had no dissociative effect. Guanosine 5′-triphosphate dissociated DRAG from the membrane at a concentration causing 50% dissociation (EC₅₀) of 5.0 ± 0.5 mM; guanosine disphosphate had an EC₅₀ of 15.0 ± 2.0 mM. We propose that GTP is a potential participant in the regulation of DRAG, possibly controlling the extent of DRAG association with the membrane. Received: 2 November 1998 / Accepted: 6 April 1999     

D-alpha-Hydroxyglutarate dehydrogenase of Rhodospirillum rubrum.

D-alpha-Hydroxyglutarate dehydrogenase of R. rubrum grown anaerobically in the light was partially purified and some properties were investigated. 1. The enzyme catalyze stoichiometrically the dehydrogenation reaction of D-alpha-hydroxyglutarate into alpha-oxoglutarate, coupled with the reduction of 2, 6-dichlorophenolindophenol. 2. Cytochrome c2, cytochrome c, and ferricyanide are effective as electron acceptors with the crude enzyme but not with the purified one, whereas NAD+ and NADP+ are completely ineffective. The enzyme is thought to play a role in the electron transport system of the organism. 3. D-alpha-Hydroxyglutarate is virtually the sole substrate for the enzyme. The apparent activity against L-alpha-hydroxyglutarate is presumed to be due to contamination of the L-isomer sample with the D-isomer. The enzyme shows barely detectable activity against both isomers of malate and virtually no activity against DL-lactate and glycolate. 4. Both isomers of malate and oxalate, which are presumably substrate analogues, inhibit the enzyme activity. 5. The enzyme is not an inducible enzyme but rather is a constitutive one for R. rubrum, unlike from the enzyme of Pseudomonas putida which is an inducible enzyme for the catabolism of lysine.