The regulation of ferredoxin-dependent nitrogenase activity in Rhodospirillum rubrum and Rhodopseudomonas capsulata

Abstract The regulatory properties of Rhodospirillum rubrum nitrogenase reduced by either the endogenous electron donor (ferredoxin) or an artificial donor (dithionite) were examined. The nitrogenase obtained from glutamate-grown cells required activating enzyme for maximum activity with either reductant. The activating enzyme requirement of ferredoxin-dependent nitrogenase activity implies a physiological significance of the activating enzyme in R. rubrum. Rhodopseudomonas capsulata nitrogenase also required activating enzyme when dithionite was the reductant, but there appeared to be no activating enzyme requirement with ferredoxin as the reductant. Because the catalytic activity of the enzyme was very low under these conditions, the physiological significance of activating enzyme in this organism remains in question.     

A comparison of electron transport and photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum

The photophosphorylation systems of Rhodopseudomonas capsulata and Rhodospirillum rubrum chromatophores have been compared in respect to the effects of artificial electron carries [N-methyl-phenazonium methosulfate (PMS) and diaminodurene], reducing agents (ascorbate in particular), and various quinones in the absence and presence of the electron transport inhibitors antimycin A and dibromothymoquinone (DBMIB). In addition, the effects of both inhibitors on photosynthetic electron transport through cytochromes b and c has been followed. From the results obtained, it appears that in both organisms: a) ubiquinone functions as an electron carrier between the cytochromes, and b) both antimycin A and DBMIB inhibit cyclic electron flow in the segment ... cytochrome bubiquinone»cytochrome c ..., but at different sites. The systems apparently differ mainly in respect to the nature of the electron flow by-pass shunt that is evoked in the presence of PMS; thus, in R. rubrum, PMS catalyzes a shunt that by-passes both cytochrome b and ubiquinone, whereas in Rps. capsulata the PMS shunt seems to circumvent only ubiquinone.Abbreviations BChl bacteriochlorophyll - DAD diaminodurene=2,3,5,6-tetramethyl-p-phenylenediamine - DBMIB dibromothymoquinone=2,5-dibromo-6-isopropyl-3-methylbenzoquinone - HOQNO heptylhydroxyquinoline-N-oxide - PMS N-methylphenazonium methosulfate     

Die Fettsäuren in ganzen Zellen,Thylakoiden und Lipopolysacchariden von Rhodospirillum rubrum und Rhodopseudomonas capsulata

Zusammenfassung Die photosynthetischen Bakterien Rhodospirillum rubrum und Rhodopseudomonas capsulata wurden auf ihre Fettsäurezusammensetzung untersucht. Die Hauptfettsäuren von R. rubrum waren C_16:0 (11%), C_16:1 (30%) und C_18:1 (52%). Vaccensäure (C_18:1) bildete 94% der Fettsäuren von Rps. capsulata. Anaerobe Lichtzellen (thylakoidhaltig) unterschieden sich nicht in ihrem Fettsäuremuster von aeroben Dunkelzellen (thylakoidfrei). Gereinigte Thylakoide aus Lichtzellen zeigten das gleiche Fettsäuremuster wie die ganzen Zellen.Nach Phenol/Wasser-Extraktion der ganzen Zellen bei 68° C war bei beiden Organismen sowohl aus Licht- als auch aus Dunkelzellen eine Substanz aus der gäßrigen Phase isolierbar, welche in den Sedimentationseigenschaften mit den Lipopolysacchariden der Enterobacteriaceae übereinstimmte und nach orientierenden Untersuchungen Zucker enthält. Aus ihr wurde ein Fettsäuregemisch gewonnen, dessen Zusammensetzung von dem aus ganzen Zellen erheblich abwich. In Rps. capsulata enthielt es C_12:1 (40%) und C_16:0 (50%), während in R. rubrum sich das Fettsäuremuster über den Bereich von C₁₀ bis C₂₀ erstreckte. Licht- und Dunkelzellen wiesen in dieser Substanz Unterschiede in der Fettsäurezusammensetzung auf. Der quantitative Anteil der Fettsäuren in dieser Substanz, bezogen auf die Gesamtfettsäuren der Zelle, betrug in Licht- und Dunkelzellen 5–7%. Hydroxy-myristinsäure ließ sich in beiden Organismen nicht nachweisen.

---

Fatty acid composition of whole cells, thylakoids and lipopolysaccharides of Rhodospirillum rubrum and Rhodopseudomonas capsulata

Summary The fatty acid composition of the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata was investigated. The bulk of fatty acids of R. rubrum consisted of C_16:0 (11%), C_16:1 (30%), and C_18:1 (52%). The major fatty acid of Rps. capsulata was vaccenic acid (C_18:1), which accounted for 94% of the total fatty acids. Cells of both organisms, which were grown anaerobically in the light and fitted out with thylakoids had the same fatty acid composition as cells grown aerobically in the dark, which have no thylakoids.Purified thylakoids had the same fatty acid pattern as whole cells. Whole cells of light and dark cultures were extracted with phenol/water at 68° C. An opalizing fraction in the aqueous phase was sedimentable in the ultracentrifuge like the lipopolysaccharides of the Enterobacteriaceae. The pattern of fatty acids in this compound differed considerably from that of whole cells. The major fatty acids in this macromolecular fraction were C_12:1 (40%) and C_16:0 (50%) in Rps. capsulata, whereas in R. rubrum the whole range of fatty acids from C₁₀ to C₂₀ was demonstrable. Light and dark grown cells differed in the fatty acid composition of that compound. The fatty acid content of the extracted fraction accounted for 5–7% of the total fatty acids of whole cells. No hydroxymyristic acid could be identified in either R. rubrum or Rps. capsulata.

     

Manganese, an Essential Trace Element for N2 Fixation by Rhodospirillum rubrum and Rhodopseudomonas capsulata: Role in Nitrogenase Regulation

Nitrogenase (N(2)ase) from the photosynthetic bacterium Rhodospirillum rubrum can exist in two forms, an unregulated form (N(2)ase A) and a regulatory form (N(2)ase R), the latter being identified in vitro by its need for activation by a Mn(2+)-dependent N(2)ase activating system. The physiological significance of this Mn(2+)-dependent N(2)ase activating system was suggested here by observations that growth of R. rubrum and Rhodopseudomonas capsulata on N(2) gas (a condition that produces active N(2)ase R) required Mn(2+), but growth on ammonia or glutamate did not. Manganese could not be shown to be required for the biosynthesis of either nitrogenase or glutamine synthetase or for glutamine synthetase turnover, but it was required for the in vitro activation of N(2)ases from N(2) and glutamate-grown R. rubrum and R. capsulata cells. Chromatium N(2)ase, in contrast, was always fully active and did not require Mn(2+) activation, suggesting that only the purple nonsulfur bacteria are capable of controlling their N(2)ase activity by this new type of regulatory system. Although R. rubrum could not substitute Fe(2+) for Mn(2+) in the in vivo N(2) fixation process, Fe(2+) and, to a lesser extent, Co(2+) could substitute for Mn(2+) in the in vitro activation of N(2)ase. Electron paramagnetic resonance spectroscopy of buffer-washed R. rubrum chromatophores showed lines characteristic of Mn(2+). Removal of the Mn(2+)-dependent N(2)ase activating factor by a salt wash of the chromatophores removed 90% of the Mn(2+), which suggested a specific coupling of this metal to the activating factor. The data presented here all indicate that Mn(2+) plays an important physiological role in regulating the N(2) fixation process by these photosynthetic bacteria.     

Nanosecond fluorescence from chromatophores of Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Single-photon counting techniques were used to measure the fluorescence decay from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum chromatophores after excitation with a 25-ps, 600-nm laser pulse. Electron transfer was blocked beyond the initial radical-pair state (PF) by chemical reduction of the quinone that serves as the next electron acceptor. Under these conditions, the fluorescence decays with multiphasic kinetics and at least three exponential decay components are required to describe the delayed fluorescence. Weak magnetic fields cause a small increase in the decay time of the longest component. The components of the delayed fluorescence are similar to those found previously with isolated reaction centers. We interpret the multi-exponential decay in terms of two small (0.01-0.02 eV) relaxations in the free energy of PF, as suggested previously for reaction centers. From the initial amplitudes of the delayed fluorescence, it is possible to calculate the standard free-energy difference between the earliest resolved form of PF and the excited singlet state of the antenna complexes in R. rubrum strains S1 and G9. The free-energy gap is found to be about 0.10 eV. It also is possible to calculate the standard free-energy difference between PF and the excited singlet state of the reaction center bacteriochlorophyll dimer (P). Values of 0.17 to 0.19 eV were found in both R. rubrum strains and also in Rps. sphaeroides strain 2.4.1. This free-energy gap agrees well with the standard free-energy difference between PF and P determined previously for reaction centers isolated from Rps. sphaeroides strain R26. The temperature dependence of the delayed fluorescence amplitudes between 180 K and 295 K is qualitatively different in isolated reaction centers and chromatophores. However, the temperature dependence of the calculated standard free-energy difference between P* and PF is similar in reaction centers and chromatophores of Rps. sphaeroides. The different temperature dependence of the fluorescence amplitudes in reaction centers and chromatophores arises because the free-energy difference between P* and the excited antenna is dominated by the entropy change associated with delocalization of the excitation in the antenna. We conclude that the state PF is similar in isolated reaction centers and in the intact photosynthetic membrane. Chromatophores from Rps. sphaeroides strain R-26 exhibit an anomalous fluorescence component that could reflect heterogeneity in their antenna.     

Thermostability of Rhodopseudomonas viridis and Rhodospirillum rubrum chromatophores reflecting physiological conditions

Relationships between growth conditions and thermostability were examined for photosynthetic inner membranes (chromatophores) from Rhodopseudomonas viridis and Rhodospirillum rubrum of which morphology, lipid composition, and protein/lipid rate are rather mutually different. Signals observed by differential scanning calorimetry of the chromatophores were correlated with thermal state transitions of the membrane components by reference to temperature dependencies of circular dichroism and absorption spectra of the purified supramolecule comprising a photoreaction center and surrounding light-harvesting pigment-protein complexes that are the prominent proteins in both membranes. The differential scanning calorimetry curves of those chromatophores exhibited different dependencies on growth stages and environmental temperatures. The obtained result appeared to reflect the differences in the protein/lipid rate and protein-lipid specificity between the two chromatophores.     

Preliminary crystallographic data for cytochromes c' of Rhodopseudomonas capsulata and Rhodospirillum molischianum.

Crystallization conditions and unit cell parameters are reported for cytochromes c′ of Rhodopseudomonas capsulata and Rhodospirillum molischianum. While both proteins naturally occur as dimers having identical subunits of M_r ~ 14,000, R. capsulata was found to crystallize in the hexagonal space group P6₂ (or its enantiomorph P6₄) with one subunit per crystallographic asymmetric unit. This result suggests that the subunits of this molecule are related by exact 2-fold symmetry.     

Propionate formation in Rhodospirillum rubrum under anaerobic dark conditions

Experiments with 14C labelled propionyl-CoA, methylmalonyl-CoA and succinyl-CoA showed that these compounds are intermediates of propionate synthesis in fermentative metabolism of Rhodospirillum rubrum. The rate of propionate and succinate production is dependent on the CO2 concentration of the medium. There is, however, no evidence for a transcarboxylation, and high concentrations of propionate in the medium did not inhibit propionate synthesis as in the case in propionibacteria. PEP-carboxykinase (EC 4.1.1.32) and propionyl-CoA-carboxylase (EC 6.4.1.3) showed high activities, whereas the other two PEP-carboxylases (EC 4.1.1.31, EC 4.1.1.38), and the pyruvate-carboxylase (EC 4.1.1.1.) showed only very low activity. It is probable that in pyruvate fermentation metabolism of R. rubrum no specific enzymes are activated for propionate formation and all enzymes are still present from aerobic or phototrophic preculture.     

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     

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.     

Urea: An intermediate of aerobic and anaerobic purine degradation in Rhodopseudomonas capsulata

Abstract A mutant strain ( pur ^-) defective in utilization of purines was isolated from Rhodopseudomonas capsulata . In the mutant, the loss of purine utilization correlated with urease deficiency. In contrast to the wild-type strain, the mutant catalyzed release of urea from purines. The nitrogen of the purine ring was completely liberated as urea indicating that the latter compound is an intermediate of the purine degradation pathway in Rps. capsulata . The degradation pattern was identical under aerobic and anaerobic conditions.     

Characterization of Rhodopseudomonas capsulata

Thirty-three strains of Rhodopseudomonas capsulata have been studied in order to develop a more comprehensive characterization of the species. On the basis of morphological, nutritional, physiological and other properties, the characteristics of an ideal biotype have been defined, which can be used to distinguish Rps. capsulata from similar purple bacteria. In this connection, two properties of Rps. capsulata are of particular note: a) sensitivity to penicillin G is 10³–10⁵ times greater than that shown by closely related species, and b) all strains examined are susceptible to lysis by one or more strains of host species-specific virulent bacteriophages. It appears that members of the species Rps. capsulata form a stringent taxonomic grouping.     

Further studies on ribulose-l,5-diphosphate carboxylase from Rhodopseudomonas spheroides and Rhodospirillum rubrum

Some enzymic properties of RuDP carboxylase isolated from Rhodopseudomonasspheroides and Rhodospirillum rubrum, grown under autotrophic,semi-autotrophic and heterotrophic conditions were studied.Regardless of the growing condition, the nature of the enzymefrom each respective bacterium remained unchanged. The molecularweight of R. spheroides and R. rubrum RuDP carboxylase was estimatedto be approximately 2.4x10⁵ and 8.3x10⁴, respectively. Bothenzymes require Mg⁺⁺. Their kinetic properties were also examined. ¹Part XII. Structure and Function of Chloroplast Proteins ²Supported in part by research grants from the Ministry of Educationof Japan (No. 8719), USPHS (AM. 10792-03) and the Asahi Press(Tokyo) (Received March 18, 1970; )     

Growth and adaptation to phototrophic conditions of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides at different temperatures

The influence of temperature on yields of cell protein and bacteriochlorophyll as well as on the rates of growth and bacteriochlorophyll synthesis was studied with Rhodospirillum rubrum and Rhodopseudomonas sphaeroides. Under chemotrophic conditions net cell-protein production increased in cultures of both species along with temperature from 14°C up to the optimum at 33°C. Under phototrophic conditions cell-protein yields were largely constant within the range from 21°C to 33°C. At temperatures below 21°C and above 33°C yields decreased. These results are interpreted in terms of coupling between energy yielding or redox equivalent providing metabolisms and cell biosynthesis. Upon adaptation from chemotrophic to phototrophic conditions a direct relationship between temperature increase and bacteriochlorophyll level was observed. Arrhenius plots of both, specific growth rates and rates of bacteriochlorophyll synthesis, revealed discontinuities at about 20°C. Temperature coefficients either above or below those discontinuities were similar in both species. In R. rubrum temperature coefficients of the synthesis of total bacteriochlorophyll were also representative of the synthesis of photochemical reaction center and light harvesting bacteriochlorophylls. But in R. sphaeroides significant differences were observed between temperature coefficients of the syntheses of bacteriochlorophylls of the costantly composed reaction centerlight harvesting complex on one hand and of both, total and the quantitatively variable light harvesting bacteriochlorophylls on the other. The results are interpreted in light of hypotheses on the regulation (a) of cellular bacteriochlorophyll levels as well as (b) of the ratio of functionally different bacteriochlorophylls in the photosynthetic apparatus.Abbreviation Bchl bacteriochlorophyll     

Isolation of cytochrome bc 1 complexes from the photosynthetic bacteria Rhodopseudomonas viridis and Rhodospirillum rubrum

Cytochrome bc ₁ complexes have been isolated from wild type Rhodopseudomonas viridis and Rhodospirillum rubrum and purified by affinity chromatography on cytochrome c-Sepharose 4B. Both complexes are largely free of bacteriochlorophyll and carotenoids and contain cytochromes b and c ₁ in a 2:1 molar ratio. For the Rps. viridis complex, evidence has been obtained for two spectrally distinct b-cytochromes. The R. rubrum complex contains a Rieske iron-sulfur protein (present in approximately 1:1 molar ratio to cytochrome c ₁) and catalyzes an antimycin A- and myxothiazol-sensitive electron transfer from duroquinol to equine cytochrome c or R. rubrum cytochrome c ₂. Although an attempt to prepare a cytochrome bc ₁ complex from the gliding green bacterium Chloroflexus aurantiacus was not successful, membranes isolated from phototrophically grown Cfl. aurantiacus were shown to contain a Rieske iron-sulfur protein and protoheme (the prosthetic group of b-type cytochromes).Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.